Chemically modified small molecules

ABSTRACT

Methods of modifying the rate of systemic absorption of a drug administered to a subject by a pulmonary route, the method comprising covalently conjugating a hydrophilic polymer to a drug, wherein the drug has a half-life of elimination from the lung of less than about 180 minutes, to form a drug-polymer conjugate, wherein the drug-polymer conjugate has a net hydrophilic character and a weight average molecular weight of from about 50 to about 20,000 Daltons, and wherein the half-life of elimination from the lung of the drug-polymer conjugate is at least about 1.5-fold greater than the half-life of elimination from the lung of the drug, wherein the half-life of elimination from the lung is measured by bronchoalveolar lavage followed by assaying residual lung material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/271,158, filed Oct. 11, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/710,167, filed Feb. 22, 2010, now U.S. Pat. No.8,067,431, which is a continuation of U.S. patent application Ser. No.11/344,404, filed Jan. 30, 2006, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 11/015,196,filed Dec. 16, 2004, now U.S. Pat. No. 7,786,133, which claims priorityto U.S. Provisional Patent Application No. 60/530,122, filed Dec. 16,2003, all five applications of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

This invention provides chemically modified small molecules and relatedmethods that possess certain advantages over small molecules lacking thechemical modification. The chemically modified small molecules describedherein relate to and/or have application(s) in the fields of drugdiscovery, pharmacotherapy, physiology, organic chemistry, polymerchemistry, and others.

BACKGROUND OF THE INVENTION

The use of proteins as active agents has expanded in recent years due toseveral factors: improved techniques for identifying, isolating,purifying and/or recombinantly producing proteins; increasedunderstanding of the roles of proteins in vivo due to the emergence ofproteonomics; and improved formulations, delivery vehicles andapproaches for chemically modifying proteins to enhance theirpharmacokinetic or phamacodynamic properties. With respect to improvedapproaches for chemically modifying proteins, covalent attachment of apolymer such as poly(ethylene glycol) or PEG to a protein has been usedto improve the circulating half-life, decrease immunogenicity, and/orreduce proteolytic degradation. This approach of covalently attachingPEG to a protein or other active agent is commonly referred to asPEGylation. Proteins for injection that are modified by covalentattachment of PEGs are typically modified by attachment of relativelyhigh molecular weight PEG polymers that often range from about 5,000 toabout 40,000 Daltons.

While modification of relatively large proteins for the purpose ofimproving their pharmaceutical utility is perhaps one of the most commonapplications of PEGylation, PEGylation has also been used, albeit to alimited degree, to improve the bioavailability and ease of formulationof small molecule drugs having poor aqueous solubilities. For instance,water-soluble polymers such as PEG have been covalently attached toartilinic acid to improve its aqueous solubility. See, for example, U.S.Pat. No. 6,461,603. Similarly, PEG has been covalently attached totriazine-based compounds such as trimelamol to improve their solubilityin water and enhance their chemical stability. See, for example,International Patent Publication WO 02/043772. Covalent attachment ofPEG to bisindolyl maleimides has been employed to improve poorbioavailability of such compounds due to low aqueous solubility. See,for example, International Patent Publication WO 03/037384. PEG chainsattached to small molecule drugs for the purpose of increasing theiraqueous solubility are typically of sizes ranging from about 500 Daltonsto about 5000 Daltons, depending upon the molecular weight of the smallmolecule drug.

Active agents can be dosed by any of a number of administration routesincluding injection, oral, inhalation, nasal, and transdermal. One ofthe most preferred routes of administration, due to its ease, is oraladministration. Oral administration, most common for small moleculedrugs (i.e., non-protein-based drugs), is convenient and often resultsin greater patient compliance when compared to other routes ofadministration. Unfortunately, many small molecule drugs possessproperties (e.g., low oral bioavailability) that render oraladministration impractical. Often, the properties of small moleculedrugs that are required for dissolution and selective diffusion throughvarious biological membranes directly conflict with the propertiesrequired for optimal target affinity and administration. The primarybiological membranes that restrict entrance of small molecule drugs intocertain organs or tissues are membranes associated with certainphysiological barriers, e.g., the blood-brain barrier, theblood-placental barrier, and the blood-testes barrier.

The blood-brain barrier protects the brain from most toxicants.Specialized cells called astrocytes possess many small branches, whichform a barrier between the capillary endothelium and the neurons of thebrain. Lipids in the astrocyte cell walls and very tight junctionsbetween adjacent endothelial cells limit the passage of water-solublemolecules. Although the blood-brain barrier does allow for the passageof essential nutrients, the barrier is effective at eliminating thepassage of some foreign substances and can decrease the rate at whichother substances cross into brain tissue.

The placental barrier protects the developing and sensitive fetus frommany toxicants that may be present in the maternal circulation. Thisbarrier consists of several cell layers between the maternal and fetalcirculatory vessels in the placenta. Lipids in the cell membranes limitthe diffusion of water-soluble toxicants. Other substances such asnutrients, gases, and wastes of the developing fetus can, however, passthrough the placental barrier. As in the case of the blood-brainbarrier, the placental barrier is not totally impenetrable buteffectively slows down the diffusion of many toxicants from the motherto the fetus in the art.

For many orally administered drugs, permeation across certain biologicalmembranes such as the blood-brain barrier or the blood-placental barrieris highly undesirable and can result in serious side-effects such asneurotoxicity, insomnia, headache, confusion, nightmares orteratogenicity. These side effects, when severe, can be sufficient tohalt the development of drugs exhibiting such undesirable brain orplacental uptake.

U.S. Published Application No. 2003/0161791 A1, published Aug. 28, 2003,discloses water-soluble polymer conjugates of retinoic acid. Theconjugates are prepared by covalent attachment of a water-solublepolymer such as polyethylene glycol to a retinoid such as retinoic acid.The conjugates are useful for inhalation therapy of conditions of therespiratory tract.

Thus, there is a need for new methods for effectively delivering drugs,and in particular small molecule drugs, to a patient whilesimultaneously reducing the adverse and often toxic side-effects ofsmall molecule drugs. Specifically, there is a need for improved methodsfor delivering drugs that possess an optimal balance of good oralbioavailability, bioactivity, and pharmacokinetic profile. The presentinvention meets this and other needs.

SUMMARY OF THE INVENTION

The invention provides methods of modifying the rate of systemicabsorption of a drug administered to a subject by a pulmonary route, themethod comprising covalently conjugating a hydrophilic polymer to adrug, wherein the unconjugated drug has a half-life of elimination fromthe lung of less than about 180 minutes, to form a drug-polymerconjugate, wherein the drug-polymer conjugate has a net hydrophiliccharacter and a weight average molecular weight of from about 50 toabout 20,000 Daltons, and wherein the half-life of elimination from thelung of the drug-polymer conjugate is at least about 1.5-fold greaterthan the half-life of elimination from the lung of the unconjugateddrug. In some embodiments, the half-life of elimination from the lung ofthe drug-polymer conjugate is at least about 2-fold, 4-fold, 10-fold,20-fold, 50-fold, 100-fold, or 500-fold greater than the half-life ofelimination from the lung of the unconjugated drug.

In some embodiments, the hydrophilic polymer comprises a polymer chosenfrom polyethylene glycols and polyethylene oxides. In some embodiments,the weight average molecular weight of the polymer is from about 1000 toabout 3500 Daltons. In some embodiments, the drug has a molecular weightof less than about 1500.

The hydrophilic polymer may comprise a polyethylene glycol, includingfor example, linear polyethylene glycols, branched polyethylene glycols,forked polyethylene glycols, and dumbbell polyethylene glycols. In someembodiments, the hydrophilic polymer comprises a polymer from apolydisperse population. In some embodiments, the hydrophilic polymer isa polymer chosen from monodisperse, bimodal, trimodal, or tetramodalpolymer populations.

The invention also provides methods of controlling the lung residencetime of a drug pulmonarily administered, comprising covalently attachingto the drug a hydrophilic polymer molecule having a weight averagemolecular weight of from about 50 to about 4000 Daltons, to form adrug-polymer conjugate. The hydrophilic polymer may be polyethyleneglycol. The weight average molecular weight of the hydrophilic polymeris, in some embodiments, from about 1000 to about 3500 Daltons. In someembodiments, the drug has a molecular weight of less than about 1500. Insome embodiments, the drug-polymer conjugate exhibits a net hydrophiliccharacter.

The invention also provides methods of controlling the rate of systemicabsorption of a drug pulmonarily administered comprising covalentlyattaching to the drug a hydrophilic polymer molecule having a weightaverage molecular weight of from about 50 to about 4000 Daltons, to forma drug-polymer conjugate.

The invention also provides pharmaceutical compounds for pulmonaryadministration comprising a drug covalently attached to a hydrophilicpolymer, wherein the pharmaceutical compound has a net hydrophiliccharacter, and wherein the weight average molecular weight of thehydrophilic polymer is from about 50 to about 3500 Daltons. In someembodiments, the invention provides compositions comprising thepharmaceutical compound according to the invention, and at least onepharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is in liquid form, and in some embodiments,in dry form. The invention also provides aerosols comprising thecomposition according to the invention. The invention also providesinhaler devices including the compositions of the invention. In someembodiments, the compositions comprise particles having a mass medianaerodynamic diameter (MMAD) of less than about 10 microns, or less thanabout 5 microns. In some embodiments, the composition comprises a drypowder. In some embodiments, the inhalers of the invention arecharacterized by an emitted dose of at least about 30 percent. Theinvention also provides spray-dried compositions.

In some embodiments of the invention, the hydrophilic polymer iscovalently attached to the active ingredient molecule by ahydrolytically unstable linkage, which can be a linkage chosen fromester, thioester, and amide. In some embodiments, the hydrophilicpolymer is covalently attached to the active ingredient by ahydrolytically stable linkage. In some embodiments, the hydrophilicpolymer is polyethylene glycol, which may be chosen from linearpolyethylene glycols, branched polyethylene glycols, forked polyethyleneglycols, and dumbbell polyethylene glycols. The invention also providesunit dosage forms comprising the compositions according to theinvention.

The invention also provides compounds according to the invention,wherein the weight average molecular weight of the hydrophilic polymeris from about 50 to about 3500 Daltons and wherein pulmonaryadministration of the compound results in a half-life of eliminationfrom the lung that can be described by the equation,t_(1/2)-el=12.84*(1-e^(−kMW)), where k=0.000357, MW=molecular weight inDaltons, and t_(1/2)=elimination half-life in hours. The invention alsoprovides methods of treating a systemic disease comprising pulmonarilyadministering the compounds according to the invention.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent when read in conjunction withthe following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of plasma concentration versus time for 13-cis retinoicacid (“13-cis-RA”) and exemplary small PEG conjugates thereof(PEG₃-13-cis retinamide, “PEG₃-13-cis RA”; PEG₅-13-cis retinamide,“PEG₅-13-cis RA; PEG₇-13-cis retinamide, “PEG₇-13-cis RA; andPEG₁₁-13-cis retinamide, “PEG₁₁-13-cis RA”) administered to SpragueDawley rats as described in detail in Example 7.

FIG. 2 is a plot of plasma concentration versus time for 6-naloxol andexemplary small PEG conjugates thereof (3-mer, 5-mer, 7-mer)administered to Sprague Dawley rats as described in detail in Example 7.

FIG. 3 is a plot demonstrating the effect PEG chain length on theintestinal transport (as an indicator of oral bioavailability) ofvarious PEG-13-cis-RA conjugates and 13-cis-RA in Sprague-Dawley rats.

FIG. 4 is a plot demonstrating the effect of covalent attachment ofvarious sized PEG-mers on the blood-brain barrier transport of 13-cis-RAand various PEG-13-cis-RA conjugates.

FIG. 5 is a plot demonstrating the effect of covalent attachment ofvarious sized PEG-mers on the intestinal transport (as an indicator oforal bioavailability) of naloxone and PEG_(n)-Nal.

FIG. 6 is a plot showing the effect of covalent attachment of varioussized PEG-mers on the blood-brain barrier transport of naloxone andPEG_(n)-Nal.

FIG. 7 is a plot demonstrating the pharmacokinetics of naloxone andPEG_(n)-Nal in rats following oral gavage.

FIG. 8 and FIG. 9 are plots demonstrating the effect of covalentattachment of various sized PEG-mers on the level of naloxonemetabolites and PEG_(n)-Nal metabolites.

FIG. 10 is mass spectrum of methoxy-PEG-350 obtained from a commercialsource (Sigma-Aldrich). As can be seen from the analysis, although thereagent is sold as methoxy-PEG having a molecular weight of 350, thereagent is actually a mixture of nine distinct PEG oligomers, with thenumber of monomer subunits ranging from approximately 7 to approximately15.

FIG. 11 diagrammatically shows the experimental strategy of Example 12.

FIG. 12 shows the elimination of PEG-FITC from BAL.

FIG. 13 shows the relationship between MW and Elimination half-life fromBAL for a PEG-FITC conjugate.

FIG. 14 shows the uptake of PEG-FITC into BAL cells.

FIG. 15 shows the percentage dose of PEG-FITC that associates with thecellular fraction of BAL.

FIG. 16 shows the association of PEG-FITC with residual lung material.

FIG. 17 shows the concentration of PEG-FITC in serum.

FIGS. 18A and 18B show the total mass recovered in lung-derivedfractions.

FIGS. 19A and 19B show the elimination of PEG-FITC from all combinedlung compartments.

FIG. 20 shows in vitro permeability for Calu-3 cell studies.

FIG. 21 illustrates cell-based permeability plotted versus the in vivoabsorption rate.

FIG. 22 shows the relationship between log P and PEG size for PEG-FITCconjugates.

FIG. 23 shows elimination from the lung for 2K PEG and 2K PEG-FITC.

FIG. 24 shows that increasing the dose of 2K PEG 10-fold does notsignificantly alter the elimination rate from the lung.

FIG. 25 shows the rate of disappearance from the lung of CIPRO andPEG-CIPRO conjugates.

FIG. 26 shows the rate in appearance of the plasma of CIPRO andPEG-CIPRO.

FIG. 27 shows the rate of appearance in the plasma of CIPRO.

FIG. 28 shows steroid-induced nuclear translocation assay of GR in CHOcells.

FIG. 29 shows the activation of Luciferase by TAA, PEG-3-TA, andPEG-7-TA.

FIG. 30 shows the time course activation of Luciferase by TAA.

FIG. 31 shows the difference in binding affinity between TA and thePEG-TA derivatives.

DETAILED DESCRIPTION OF THE INVENTION

It must be noted that, as used in this specification, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. In describing and claiming the present invention,the following terminology will be used in accordance with thedefinitions described below.

“Water soluble” as in a “water-soluble oligomer” indicates an oligomerthat is at least 35% (by weight) soluble, preferably greater than 95%soluble, and more preferably greater than 99% soluble, in water at roomtemperature at physiological pH (about 7.2-7.6). Typically, anunfiltered aqueous preparation of a “water-soluble” oligomer transmitsat least 75%, more preferably at least 95%, of the amount of lighttransmitted by the same solution after filtering. On a weight basis, a“water soluble” oligomer is preferably at least 35% (by weight) solublein water, more preferably at least 50% (by weight) soluble in water,still more preferably at least 70% (by weight) soluble in water, andstill more preferably at least 85% (by weight) soluble in water. It ismost preferred, however, that the water-soluble oligomer is at least 95%(by weight) soluble in water or completely soluble in water.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, this is definedas a structural repeating unit of the oligomer. In the case of aco-oligomer, a monomeric unit is more usefully defined as the residue ofa monomer that was oligomerized to form the oligomer, since thestructural repeating unit can include more than one type of monomericunit. Preferred oligomers of the invention are homo-oligomers.

An “oligomer” is a molecule possessing from about 1 to about 30monomers. The architecture of an oligomer can vary. Specific oligomersfor use in the invention include those having a variety of geometriessuch as linear, branched, or forked, to be described in greater detailbelow. An oligomer is a type of polymer.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which all of themonomer subunits are ethylene oxide subunits. Typically, substantiallyall, or all, monomeric subunits are ethylene oxide subunits, though theoligomer may contain distinct end capping moieties or functional groups,e.g. for conjugation. Typically, PEG oligomers for use in the presentinvention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)-” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG oligomers of theinvention, the variable (n) ranges from 1 to 30, and the terminal groupsand architecture of the overall PEG can vary. When PEG further comprisesa functional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer, does notresult in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxidelinkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

An “end capping group” is generally a non-reactive carbon-containinggroup attached to a terminal oxygen of a PEG oligomer. For the purposesof the present invention, preferred are capping groups having relativelylow molecular weights such as methyl or ethyl. The end-capping group canalso comprise a detectable label. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric labels (e.g., dyes), metal ions, and radioactivemoieties.

“Branched”, in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymer “arms”extending from a branch point.

“Forked” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions effective to produce a desired reactionin the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof is meant to encompass protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater will depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, amines, and the like. Generally, a hydrolyticallystable linkage is one that exhibits a rate of hydrolysis of less thanabout 1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, single(i.e., the same) molecular weight and defined number of monomers, asdetermined by chromatography or mass spectrometry. Monodisperse oligomercompositions are in one sense pure, that is, substantially having asingle and definable number (as a whole number) of monomers rather thana large distribution. A monodisperse oligomer composition of theinvention possesses a MW/Mn value of 1.0005 or less, and morepreferably, a MW/Mn value of 1.0000. By extension, a compositioncomprised of monodisperse conjugates means that substantially alloligomers of all conjugates in the composition have a single anddefinable number (as a whole number) of monomers rather than a largedistribution and would possess a MW/Mn value of 1.0005 or less, and morepreferably, a MW/Mn value of 1.0000 if the oligomer were not attached tothe moiety derived from a small molecule drug. A composition comprisedof monodisperse conjugates can, however, include one or morenonconjugate substances such as solvents, reagents, excipients, and soforth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is symmetric about its mean, although the size of the two peaks maydiffer. Ideally, the polydispersity index of each peak in the bimodaldistribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000. By extension, a composition comprised of bimodal conjugatesmeans that substantially all oligomers of all conjugates in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution and would possessa MW/Mn value of 1.01 or less, more preferably 1.001 or less and evenmore preferably 1.0005 or less, and most preferably a MW/Mn value of1.0000 if the oligomer were not attached to the moiety derived from asmall molecule drug. A composition comprised of bimodal conjugates can,however, include one or more nonconjugated substances such as solvents,reagents, excipients, and so forth.

“Polydisperse” in reference to a polymer, refers to a composition havinga polymer present in a distribution of molecular weights. Thedistribution generally will be a normal distribution, i.e., one that hasa higher concentration of polymers with molecular weights near the mean,with a decrease in frequency as the difference from the mean molecularweight increases. The distribution may be a Gaussian distribution.

A “drug” is broadly used herein to refer to an organic, inorganic, ororganometallic compound typically having a molecular weight of less thanabout 1500. Drugs of the invention encompass oligopeptides and otherbiomolecules having a molecular weight of less than about 1500. Peptidedrugs of the invention have a molecular weight of less than about 1500Daltons. It will be understood that the term “drug” refers to any drugin its active form, any prodrug, and any active ingredient. “Drug” asused herein includes any agent, compound, composition of matter ormixture which provides some pharmacologic, often beneficial, effect thatcan be demonstrated in vivo or in vitro. This includes foods, foodsupplements, nutrients, nutriceuticals, drugs, vaccines, antibodies,vitamins, and other beneficial agents. As used herein, these termsfurther include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.“Small molecule,” “small molecule drug,” and “drug” are usedinterchangeably herein.

The terms “moiety derived from a small molecule drug” and “smallmolecule drug moiety” are used interchangeably herein to refer to theportion or residue of the parent small molecule drug up to the covalentlinkage resulting from covalent attachment of the drug (or an activatedor chemically modified form thereof) to an oligomer of the invention.

A “biological membrane” is any membrane, typically made from specializedcells or tissues, that serves as a barrier to at least some xenobioticsor otherwise undesirable materials. As used herein a “biologicalmembrane” includes those membranes that are associated withphysiological protective barriers including, for example: theblood-brain barrier; the blood-cerebrospinal fluid barrier; theblood-placental barrier; the blood-milk barrier; the blood-testesbarrier; and mucosal barriers including the vaginal mucosa, urethralmucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa,and so forth). Unless the context clearly dictates otherwise, the term“biological membrane” does not include those membranes associated withthe middle gastro-intestinal tract (e.g., stomach and small intestines).

A “biological membrane crossing rate,” as used herein, provides ameasure of a compound's ability to cross a biological barrier, such asthe blood-brain barrier (“BBB”). A variety of methods can be used toassess transport of a molecule across any given biological membrane.Methods to assess the biological membrane crossing rate associated withany given biological barrier (e.g., the blood-cerebrospinal fluidbarrier, the blood-placental barrier, the blood-milk barrier, theintestinal barrier, and so forth), are known, described herein and/or inthe relevant literature, and/or can be determined by one of ordinaryskill in the art.

A compound that “crosses the blood-brain barrier” in accordance with theinvention is one that crosses the BBB at a rate greater than that ofatenolol using the methods as described herein.

A “reduced rate of metabolism” in reference to the present invention,refers to a measurable reduction in the rate of metabolism of awater-soluble oligomer-small molecule drug conjugate as compared to rateof metabolism of the small molecule drug not attached to thewater-soluble oligomer (i.e., the small molecule drug itself) or areference standard material. In the special case of “reduced first passrate of metabolism,” the same “reduced rate of metabolism” is requiredexcept that the small molecule drug (or reference standard material) andthe corresponding conjugate are administered orally. Orally administereddrugs are absorbed from the gastro-intestinal tract into the portalcirculation and must pass through the liver prior to reaching thesystemic circulation. Because the liver is the primary site of drugmetabolism or biotransformation, a substantial amount of drug can bemetabolized before it ever reaches the systemic circulation. The degreeof first pass metabolism, and thus, any reduction thereof, can bemeasured by a number of different approaches. For instance, animal bloodsamples can be collected at timed intervals and the plasma or serumanalyzed by liquid chromatography/mass spectrometry for metabolitelevels. Other techniques for measuring a “reduced rate of metabolism”associated with the first pass metabolism and other metabolic processesare known, described herein and/or in the relevant literature, and/orcan be determined by one of ordinary skill in the art. Preferably, aconjugate of the invention can provide a reduced rate of metabolismreduction satisfying at least one of the following values: at leastabout 5%, at least about 10%, at least about 15%; least about 20%; atleast about 25%; at least about 30%; at least about 40%; at least about50%; at least about 60%; at least about 70%; at least about 80%; and atleast about 90%.

A compound (such as a small molecule drug or conjugate thereof) that is“orally bioavailable” is one that possesses a bioavailability whenadministered orally of greater than 1%, and preferably greater than 10%,where a compound's bioavailability is the fraction of administered drugthat reaches the systemic circulation in unmetabolized form.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

“Electrophile” refers to an ion, atom, or an ionic or neutral collectionof atoms having an electrophilic center, i.e., a center that is electronseeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or an ionic or neutral collectionof atoms having a nucleophilic center, i.e., a center that is seeking anelectrophilic center, and capable of reacting with an electrophile.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterobifunctional.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, typically, but not necessarily, in theform of a water-soluble oligomer-small molecule drug conjugate, andincludes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Hydrophilic” in reference to a polymer or a drug-polymer conjugatemeans a compound or conjugate having a negative octanol:water partitioncoefficient (log P).

“Hydrophilic character” means being hydrophilic.

“Hydrolytically unstable” in reference to a linker means a linker thatcan be hydrolyzed under the conditions of a body.

“Residence time” means the amount of time a substance remains in acompartment—measured by half-life of elimination from that compartment.

“Rate of systemic absorption” means the rate at which a molecule crossesan epithelial layer to enter the systemic circulation.

The present invention is directed to (among other things) compositionsof small molecule drugs that are chemically modified by covalentattachment of a water-soluble oligomer obtained from a monodisperse orbimodal composition of water-soluble oligomers. Because thewater-soluble oligomer is often obtained from a monodisperse or bimodalcomposition of water-soluble oligomers, the resulting small moleculedrug-oligomer compositions of the invention are typically exceedinglypure and well-defined from a structural standpoint.

An advantage of some of the conjugates described herein is their abilityto exhibit a reduced biological membrane crossing rate as compared tothe corresponding active agent not in conjugated form. While not wishingto be bound by theory, it is believed that molecular size is animportant factor for determining whether and to what extent any givenmolecule can pass or cross any given biological membrane. For example,most if not all protective barriers, rely at least in part on highlypacked cells that form a membrane having tight junctions through whichonly relatively small molecules can pass. Thus, for a given smallmolecule drug, the attachment of a water-soluble polymer to the smallmolecule drug provides a conjugate that is necessarily larger and withthe expectation that the conjugate will either be prevented fromcrossing a biological membrane or will have a reduced biologicalmembrane crossing rate as compared to the unconjugated small moleculedrug.

As will be shown in further detail below and in the Experimentalsection, however, reducing the rate of biological membrane crossing byincreasing molecular size by conjugating a water-soluble oligomer to asmall molecule drug does not always provide a completely satisfactoryconjugate. Often, the conjugate will be provided as a compositioncomprising monodisperse or bimodal conjugates. Again, while not wishingto be bound by theory, it is believed that even very small differencesin the number of monomers between conjugates can provide relativelylarge differences in properties such as pharmacologic activity,metabolism, oral bioavailability, biological membrane crossing rate,solubility and others.

Furthermore, as is evidenced by the mass spectrum provided in FIG. 10,commercially available oligomer compositions such as PEG-350 are, infact, relatively impure in that a range of oligomer sizes are present inthe composition. Thus, the use of such relatively impure oligomercompositions (without further purification) in the synthesis ofconjugates would result in a wide range of conjugate molecular weightsizes (as a result of the wide range of molecular weights in thecomposition used to form the conjugate). As a consequence, the resultingconjugate composition comprises many species of conjugates, wherein eachconjugate would be expected to have different properties. From aregulatory and medicinal perspective, compositions comprising moietieshaving markedly different properties are ideally avoided.

As a result, in one or more embodiments, the present invention providesconjugates that are not only relatively large (as compared to thecorresponding unconjugated small molecule drug) to reduce biologicalmembrane crossing (again, as compared to the corresponding unconjugatedsmall molecule drug), but are substantially pure as well to ensureconsistent and desired activity and other properties of the composition.Thus, a composition is often provided comprising monodisperse or bimodalconjugates, each conjugate comprised of a moiety derived from a smallmolecule drug covalently attached by a stable linkage to a water-solubleoligomer, wherein said conjugate exhibits a reduced biological membranecrossing rate as compared to the biological membrane crossing rate ofthe small molecule drug not attached to the water-soluble oligomer.

As previously indicated, use of discrete oligomers from a well-definedcomposition of oligomers to form conjugates can advantageously altercertain properties associated with the corresponding small moleculedrug. For instance, a conjugate of the invention, when administered byany of a number of suitable administration routes, such as parenteral,oral, transdermal, buccal, pulmonary, or nasal, exhibits reducedpenetration across a biological membrane (such as the biologicalmembranes associated with the blood-brain barrier and blood-placentalbarrier). It is preferred that the conjugates exhibit slowed, minimal oreffectively no crossing of biological membranes (such as the biologicalmembranes associated with the blood-brain barrier and blood-placentalbarrier), while still crossing the gastro-intestinal (GI) walls and intothe systemic circulation if oral delivery is intended. The conjugates ofthe invention maintain a degree of bioactivity as well asbioavailability in their conjugated form.

In some embodiments in which pulmonary delivery is intended, theconjugate administered may have no crossing into systemic circulation ora reduced pulmonary tissue-blood barrier crossing rate so that locallung levels are maintained for local pharmacologic activity in the lung.Indeed, it has been surprisingly discovered that there is a directrelationship between the size of the polymer conjugated to the smallmolecule and its ability to slow the absorption from the lung. In someembodiments, an exponential relationship is observed from hydrophilicpolymers, such as polyethylene glycol, having molecular weights of fromabout 50 Daltons to about 3500 Daltons. Thus, generally, as themolecular weight of the conjugated polymer increases from about 50Daltons to about 3500 Daltons, so does the half-life of absorptionthrough the lung. In some embodiments, a maximum ability to retardabsorption is achieved by hydrophilic polymers having molecular weightsof about 5000 Daltons, and additional increases in molecular weight mayhave little or no effect.

Still further, it has been surprisingly discovered that small moleculesthat are not normally readily absorbed through the lungs, can be causedto be absorbed, by their conjugation to hydrophilic polymers, accordingto the invention. While not wishing to be bound by any particular theoryof operation, it appears that the conjugation to the hydrophilic polymermay cause the small molecule to be absorbed via a paracellularmechanism, as opposed to via a transcellular operation. This phenomenonmay be a result of the imparted hydrophilicity, molecular size, or acombination of the two. Thus, small molecules that are normally taken upacross the cell membranes of epithelial cells of the lungs are,according to this possible theory of operation, taken up throughparacellular junctions. Again, for small molecules that are rapidlyabsorbed by the transcellular route, the conjugation according to theinvention often results in a slower rate of absorption. And for smallmolecules that are very slowly absorbed, or even not absorbed at all,from the lung, a relative increase in the rate of absorption is oftenimparted by the present invention.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of most solutes and drugs into the central nervous system.

Although it may be desirable for some compounds to achieve adequateconcentrations in brain tissue to pharmacologically act therein, manyother compounds that have no useful pharmacologic activity in braintissue can ultimately reach the tissues of the central nervous system.By reducing the crossing rate of entry of these non-centrally actingcompounds into the central nervous system, the risk of central nervoussystem side effects is reduced and the therapeutic effect may even beincreased.

For compounds whose degree of blood-brain barrier crossing ability isnot readily known, such ability can be determined using a suitableanimal model such as an in situ rat brain perfusion (“RBP”) model asdescribed herein. Briefly, the RBP technique involves cannulation of thecarotid artery followed by perfusion with a compound solution undercontrolled conditions, followed by a wash out phase to remove compoundremaining in the vascular space. (Such analyses can be conducted, forexample, by contract research organizations such as Absorption Systems,Exton, Pa.). More specifically, in the RBP model, a cannula is placed inthe left carotid artery and the side branches are tied off. Aphysiologic buffer containing the compound (5 micromolar) is perfused ata flow rate of 10 mL/min in a single pass perfusion experiment. After 30seconds, the perfusion is stopped and the brain vascular contents arewashed out with compound-free buffer for an additional 30 seconds. Thebrain tissue is then removed and analyzed for compound concentrationsvia liquid chromatograph with tandem mass spectrometry detection(LC/MS/MS). Alternatively, blood-brain barrier permeability can beestimated based upon a calculation of the compound's molecular polarsurface area (“PSA”), which is defined as the sum of surfacecontributions of polar atoms (usually oxygens, nitrogens and attachedhydrogens) in a molecule. The PSA has been shown to correlate withcompound transport properties such as blood-brain barrier transport.Methods for determining a compound's PSA can be found, e.g., in, Ertl,P., et al., J. Med. Chem. 2000, 43, 3714-3717; and Kelder, J., et al.,Pharm. Res. 1999, 16, 1514-1519.

A similar barrier to the blood-brain barrier is the blood-cerebrospinalfluid barrier. The blood-cerebrospinal fluid barrier creates a barrieror otherwise reduces the amount of toxic or undesirable substancesreaching the cerebrospinal fluid, which is mostly located in theventricular system and the subarachnoid space. To determine whether andto what extent a compound (e.g., a small molecule drug or conjugate)administered to a patient can cross the blood-cerebrospinal fluidbarrier, a known amount of the compound can be administered to mice byinjection. A few days following administration of the compound, samplesof mouse cerebrospinal fluid can be analyzed for the presence and amountof the compound.

The blood-placental barrier protects the developing fetus from mosttoxicants distributed in the maternal circulation. This barrier consistsof several cellular layers between the maternal and fetal circulatoryvessels in the placenta. As in the case of the blood-brain barrier, theplacental barrier is not totally impenetrable but effectively slows downthe diffusion of most toxicants. To determine whether and to what extenta compound (e.g., a small molecule drug or conjugate) administered to apregnant mammal can cross the blood-placental barrier, a known amount ofthe compound can be administered to pregnant mice by injection. A fewdays following administration of the compound, samples of mouse fetaltissue can be analyzed for the presence and amount of the compound.

The blood-milk barrier is similar to the blood-brain barrier in that abiological membrane separates and limits certain substances in thesystemic circulation from crossing through. In the case of theblood-milk barrier, the biological membrane prevents certain substancesfrom passing into the mammary glands. To determine whether and to whatextent a compound (e.g., a small molecule drug or conjugate)administered to a lactating mammal can cross the blood-milk barrier, aknown amount of the compound can be administered to lactating mice byinjection. A few days following administration of the compound, samplesof milk from the mammary glands can be analyzed for the presence andamount of the compound.

The blood-testes barrier is comprised of sustentacular cells (Sertolicells) cells which line the male reproductive tract and are joined bytight junctions. To determine whether and to what extent a compound(e.g., a small molecule drug or conjugate) administered to a male mammalcan cross the blood-testes barrier, a known amount of the compound canbe administered to male mice by injection. A few days followingadministration of the compound, the mouse's testes can be removed andanalyzed for the presence and amount of the compound.

Mucosal barriers represent another biological membrane that typicallyblocks or reduces undesirable substances from reaching systemiccirculation. Administration of a compound to the particular mucosal areaof interest and then analyzing a blood sample for the presence andamount of the compound can determine whether and to what extent thecompound crosses that particular mucosal area.

With respect to any biological membrane, the water-solubleoligomer-small molecule drug conjugate exhibits a biological membranecrossing rate that is reduced as compared to the biological membranecrossing rate of the small molecule drug not attached to thewater-soluble oligomer. Exemplary reductions in biological membranecrossing rates include reductions of: at least about 5%; at least about10%; at least about 25%; at least about 30%; at least about 40%; atleast about 50%; at least about 60%; at least about 70%; at least about80%; or at least about 90%, when compared to the biological membranecrossing rate of the small molecule drug not attached to thewater-soluble oligomer. A preferred reduction in the biological membranecrossing rate for a conjugate is at least about 20%. In some instances,it is preferred that the small molecule drug itself is one that doescross one or more of the biological membranes described herein.

The conjugates exhibiting a reduced biological membrane crossing ratewill typically comprise the structure

O—X-D

wherein: O corresponds to a water-soluble oligomer, X corresponds to astable linkage, and D corresponds to the moiety derived from a smallmolecule drug.

The moiety derived from a small molecule drug is, in one sense,different than the parent small molecule drug in that it is linked,typically through a covalent bond, to an atom that is not associatedwith the parent small molecule drug. Except for the difference of beinglinked to another atom, however, the moiety derived from a smallmolecule drug is essentially the same as the small molecule drug andwill have a similar pharmacologic mechanism of action. Thus, adiscussion of the small molecule drug serves equally well to describethe moiety derived from a small molecule drug.

The active agents used in the conjugates are small molecule drugs, thatis to say, pharmacologically active compounds typically having amolecular weight of less than about 1500 Daltons. Small molecule drugs,for the purpose of the invention, include oligopeptides,oligonucleotides, and other biomolecules having a molecular weight ofless than about 1500 Daltons. Also encompassed in the term “smallmolecule drug” is any fragment of a peptide, protein, or antibody,including native sequences and variants falling within the molecularweight range stated above.

Exemplary molecular weights of small molecule drugs include molecularweights of: less than about 1400; less than about 1300; less than about1200; less than about 1100; less than about 1000; less than about 950;less than about 900; less than about 850; less than about 800; less thanabout 750; less than about 700; less than about 650; less than about600; less than about 550; less than about 500; less than about 450; lessthan about 400; less than about 350; and less than about 300.

The small molecule drug used in the invention, if chiral, may beobtained from a racemic mixture, or an optically active form, forexample, a single optically active enantiomer, or any combination orratio of enantiomers. In addition, the small molecule drug may possessone or more geometric isomers. With respect to geometric isomers, acomposition can comprise only a single geometric isomer or a mixture oftwo or more geometric isomers. A small molecule drug for use in thepresent invention can be in its customary active form, or may possesssome degree of modification. For example, a small molecule drug may havea targeting agent, tag, or transporter attached thereto, prior to orafter covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

A small molecule for use in coupling to an oligomer of the invention maybe any of the following. Suitable agents may be selected from, forexample, respiratory drugs, anticonvulsants, muscle relaxants,anti-inflammatories, appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, bronchodilators,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, diuretics, lipidregulating agents, antiandrogenic agents, antiparasitics,anticoagulants, neoplastics, antineoplastics, hypoglycemics, nutritionalagents and supplements, growth supplements, antienteritis agents,vaccines, antibodies, diagnostic agents, and contrasting agents.

More particularly, the active agent may fall into one of a number ofstructural classes, including but not limited to small molecules,oligopeptides, polypeptides or protein mimetics, fragments, oranalogues, steroids, nucleotides, oligonucleotides, electrolytes, andthe like. Preferably, an active agent for coupling to an oligomer of theinvention possesses a free hydroxyl, carboxyl, thio, amino group, or thelike (i.e., “handle”) suitable for covalent attachment to the oligomer.Alternatively, the drug is modified by introduction of a suitable“handle”, preferably by conversion of one of its existing functionalgroups to a functional group suitable for formation of a stable covalentlinkage between the oligomer and the drug. Both approaches areillustrated in the Experimental section.

Specific examples of active agents suitable for covalent attachment toan oligomer of the invention include small molecule mimetics and activefragments (including variants) of the following: aspariginase, amdoxovir(DAPD), antide, becaplermin, calcitonins, cyanovirin, denileukindiftitox, erythropoietin (EPO), EPO agonists (e.g., peptides from about10-40 amino acids in length and comprising a particular core sequence asdescribed in WO 96/40749), dornase alpha, erythropoiesis stimulatingprotein (NESP), coagulation factors such as Factor V, Factor VII, FactorVIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, vonWillebrand factor; ceredase, cerezyme, alpha-glucosidase, collagen,cyclosporin, alpha defensins, beta defensins, exendin-4, granulocytecolony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1proteinase inhibitor, elcatonin, granulocyte macrophage colonystimulating factor (GMCSF), fibrinogen, filgrastim, growth hormoneshuman growth hormone (hGH), growth hormone releasing hormone (GHRH),GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bonemorphogenic protein-2, bone morphogenic protein-6, OP-1; acidicfibroblast growth factor, basic fibroblast growth factor, levadopa,CD-40 ligand, heparin, human serum albumin, low molecular weight heparin(LMWH), interferons such as interferon alpha, interferon beta,interferon gamma, interferon omega, interferon tau, consensusinterferon; interleukins and interleukin receptors such as interleukin-1receptor, interleukin-2, interluekin-2 fusion proteins, interleukin-1receptor antagonist, interleukin-3, interleukin-4, interleukin-4receptor, interleukin-6, interleukin-8, interleukin-12, interleukin-13receptor, interleukin-17 receptor; lactoferrin and lactoferrinfragments, luteinizing hormone releasing hormone (LHRH), insulin,pro-insulin, insulin analogues (e.g., mono-acylated insulin as describedin U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosphonates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to an oligomer of theinvention include but are not limited to amifostine, amiodarone,aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, fluconazole, voriconazole, posiconazole, goserelin,letrozole, leucovorin, levamisole, lisinopril, lovothyroxine sodium,lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, metaraminol bitartrate, methotrexate, metoclopramide,mexiletine, mitomycin, mitotane, mitoxantrone, naloxone, nicotine,nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin,pilcamycin, porfimer, prednisone, procarbazine, prochlorperazine,ondansetron, raltitrexed, sirolimus, streptozocin, tacrolimus,tamoxifen, temozolomide, teniposide, testosterone, tetrahydrocannabinol,thalidomide, thioguanine, thiotepa, topotecan, tretinoin, valrubicin,vinblastine, vincristine, vindesine, vinorelbine, dolasetron,granisetron; formoterol, fluticasone, leuprolide, midazolam, alprazolam,amphotericin B, podophylotoxins, nucleoside antivirals, aroylhydrazones, sumatriptan; macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin;aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate; polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V; penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, tiotropium,flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such aspaclitaxel; SN-38, and tyrphostines.

The above exemplary drugs are meant to encompass, where applicable,analogues, agonists, antagonists, inhibitors, isomers, polymorphs, andpharmaceutically acceptable salt forms thereof. Thus, for example, tothe extent that an exemplary drug provided above is relatively large andwould not be classified as a small molecule drug, the exemplary drug isstill listed because an analogue of that large molecule having a similaractivity but small size can be used.

Small molecule drugs particularly well suited for the invention arethose that can measurably cross a biological membrane. Small moleculedrugs exhibiting passage across the dermal barrier are alsocontemplated. In some instances, the small molecule drug is one, thatwhen administered orally or even parenterally, undesirably crosses abiological barrier to a significant degree. For example, a smallmolecule drug that undesirably crosses the blood-brain barrier is onethat exhibits a brain uptake rate greater than that of atenolol. In thisregard, small molecule drugs that have a brain uptake rate (“BUR”), whenmeasured as described herein, of greater than about 15 pmol/gm brain/secare nonlimiting examples of small molecule drugs that undesirably crossthe blood-brain barrier.

Thus, with respect to the blood-brain barrier, small molecule drugsintended for non-central nervous system indications that nonethelesscross the blood-brain barrier are preferred since conjugation of thesedrugs provides a molecule having less central nervous system sideeffects. For example, the structurally related nucleotides andnucleosides (e.g., 8-azaguanine, 6-mercaptupurine, azathioprene,thioinosinate, 6-methylthioinosinate, 6-thiouric acid, 6-thioguanine,vidarabine, cladribine, ancitabine, azacytidine,erythro-9-(2-hydroxy-3-nonyl)adenine, fludarabine, gemcitabine, and soforth) are preferred.

With respect to fludarabine, this small molecule drug exhibits about 70%oral bioavailability, and is used for treatment of chronic lymphocyticleukemia, as well as for treatment of hairy cell leukemia, non-Hodgkin'slymphoma, and mycosis fungoides. Fludarabine also exhibits centralnervous system-related side effects, with severe neurologic effectsincluding blindness, coma, and even death. Animal studies in rats andrabbits indicate that the drug may also be teratogenic. Thus, afludarabine conjugate is expected to be effective in either blocking thepenetration of drugs through the blood-brain barrier and/orblood-placenta barrier or at least slowing the crossing rate acrossthese barriers such that adverse side effects of fludarabine areameliorated.

Another class of small molecule drug that has common central nervoussystem-related side effects although is typically used for peripheralactivities is the small molecule drug class of antihistamines.Structurally, antihistamines as a class are related as aminoalkylethers. Such small molecule drugs include diphenhydramine,bromodiphenhydramine, doxylamine, carbinoxamine, clemastine,dimenhydrinate, tripelennamine, pyrilamine, methapyrilene, thonzylamine,pheniramine, chlorpheniramine, dexchlorpheniramine, bromopheniramine,dexbromopheniramine, pyrrobutamine, triprolidine, promethazine,trimeprazine, methdilazine, cyclizine, chlorcyclizine, diphenylpyraline,phenindamine, dimethindene, meclizine, buclizine, antazoline,cyproheptadine, azatadine, terfenadine, fexofenadine, astemizole,cetirizine, azelastine, azatadine, loratadine, and desloratadine.

Still another class of small molecule drug in which a reduction in theblood-brain barrier crossing rate is desired are the opioid antagonists.Opioid antagonists include, naloxone, N-methylnaloxone,6-amino-14-hydroxy-17-allylnordesomorphine, naltrendol, naltrexone,N-methylnaltrexone, nalbuphine, butorphanol, cyclazocine, pentazocine,nalmephene, naltrindole, nor-binaltorphimine, oxilorphan,6-amino-6-desoxo-naloxone, pentazocine, levallorphanmethylnaltrexone,buprenorphine, cyclorphan, levalorphan, and nalorphine, as well as thosedescribed in U.S. Pat. Nos. 5,159,081, 5,250,542, 5,270,328, and5,434,171 and in Knapp et al., “The pharmacology of Opioid Peptides” L.F. Tseng Ed., p. 15, Harwood Academic Publishers, 1995. Generally,however, any member of the oxymorphone chemical class (including theopioid antagonists above, as well as oxymorphone, codeine, oxycodone,morphine, ethylmorphine, diacetylmorphine, hydromorphone,dihydrocodeine, dihydromorphine, and methyldihydromorphine) iscontemplated.

Another chemical class of small molecule drugs are the platinumcoordination complex-based drugs. These include, for example, cisplatin,hydroplatin, carboplatin, and oxaliplatin.

Another class of small molecule drugs particularly well suited to beconjugated is the steroid class. Preferred steroids have a hydroxylgroup in their molecular structure (or an acyl group that can be reducedto form a hydroxyl group). Nonlimiting examples of steroids includealdosterone, deoxycorticosterone, fludrocortisone, cortisone,hydrocortisone, prednisolone, prednisone, medrysone, meprednisone,alclometasone, beclomethasone, betamethasone, dexamethasone,diflorasone, flumethasone, methylprednisolone, paramethasone,amcinonide, desonide, budesonide, fluocinolone, flunisolide,flurandrenolide, triamcinolone, clobetasol, halcinonide, mometasone,clocortolone, and desoximetasone.

Fluoroquinolones and related small molecule drugs in this class can beused to form conjugates. Exemplary fluoroquinolones include thoseciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin and sitafloxacin.

Still another class of drug that is generally used for peripheralindications, some members of which are known to be teratogenic, is theretinoid class of small molecule drugs. The structurally related classof retinoids include, without limitation, retinol, retinal,3-dehydroretinol, α-carotene, β-carotene, γ-carotene, δ-carotene,crytoxanthin, tretinoin, isotretinoin, etretinate, and eretin. Due tothe potential for teratogenicity for this class of small molecule drug(or any class of drug that causes teratogenicity), it is desirable toreduce potential harm to the fetus by eliminating entirely or decreasingthe rate of blood-placental barrier crossing of agents suspected ofbeing teratogens.

Additional small molecule drugs for use as part of the conjugatesdescribed herein include phenothiazines, dibenzo-diazepines,galactogugues such as metoclopramide, and thiazides. Examples ofphenothiazines include prochlorperazine, perphenazine,trifluoroperazine, and fluphenazine. Examples of dibenzo-diazepinesinclude clozapine, olanzapine, and quetiapine. Other small moleculedrugs include amlodipine, nifedipine, nimodipine, 5-hydroxytryptophan,retinoic acid, and isotretinoin. Another preferred drug is nevirapine,which readily crosses the placental barrier.

It should be noted that the small molecules referred to herein areprovided as examples only, and moreover, that the listing of these smallmolecules does not imply that the invention has the same effect on allof these molecules. Obviously, each small molecule has its ownphysical-chemical characteristics that influence its contribution to thefinal characteristics of the complex. Furthermore, the listing of all ofthese small molecules together obviously does not mean that every one isuseful for the treatment of the same condition or can be administeredvia the same route. Again, the specific qualities of the starting smallmolecule may contribute to the end use of the complex according to theinvention. For at least these reasons, it is specifically noted that thelisting of any class of small molecules, or even any small moleculewithin a class, implies the potential exclusion of that class, orspecific small molecule, from particular embodiments of the invention.Thus, for example, while reference has been made herein to retinoidssuch as retinoic acid and isotretinoin, in some embodiments, that entireclass of compounds, or specific molecules within that class, isexcluded.

Additional small molecule drugs suitable for use in the invention can befound in, for example, in “The Merck Index, 13^(th) Edition, Merck &CO., Inc. (2001); “The AHFS Drug Handbook, 2^(nd) Edition”, AmericanSociety of Health System Pharmacists and Lippincott, Williams andWilkins; “The Physicians Desk Reference”, Thomson Healthcare Inc., 2003;and “Remington: The Science and Practice of Pharmacy”, 19^(th) Edition,1995.

By modifying the small drug molecule as provided above with covalentattachment of a water-soluble oligomer obtained from a monodisperse,bimodal, or polydisperse oligomer composition, significant changes inthe small molecule drug's transport and pharmacological properties canresult. The use of a water-soluble oligomer from a monodisperse,bimodal, or polydisperse oligomer composition allows for tailoring ofdrug properties, since the resultant conjugates form a well-definedcomposition rather than a distribution of a series of small moleculedrug-oligomer conjugate species having a distribution of monomersubunits (and therefore molecular weights). As previously stated, theaddition or deletion of as little as one monomer is observed to have ameasurable effect on the properties of the resulting conjugate.Screening of a matrix of discrete oligomers of different sizes (from 1to 30 monomer subunits) can be conducted in a reasonable amount of time,and allows for the tailoring of customizing of conjugates havingoptimized properties.

The oligomers, when attached to the small molecule drug, providedifferences in properties compared to the parent small drug molecule.The use of small oligomers (in comparison to the 5K to 60K polymerchains that are typically attached to proteins) also increases thelikelihood of the drug maintaining at least a degree, and preferably asignificant degree, of its bioactivity. This feature is demonstrated inTable VI (Example 10), which provides bioactivity (EC₅₀) data forexemplary conjugates of the invention. The illustrative PEGoligomer-naloxone/naloxol conjugates possess bioactivities ranging fromabout 5% to about 35% of the unmodified parent drug, furtherdemonstrating the beneficial features of the compounds of the invention.

The oligomer typically comprises two or more monomers serially attachedto form a chain of monomers. The oligomer can be formed from a singlemonomer type (i.e., is homo-oligomeric) or two or three monomer types(i.e., is co-oligomeric). Preferably, each oligomer is a co-oligomer oftwo monomers or, more preferably, is a homo-oligomer. The monomer(s)employed result in an oligomer that is water soluble as defined herein.

Accordingly, each oligomer is typically composed of up to threedifferent monomer types selected from the group consisting of: alkyleneoxide, such as ethylene oxide or propylene oxide; olefinic alcohol, suchas vinyl alcohol, 1-propenol or 2-propenol; vinyl pyrrolidone;hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, where alkyl ispreferably methyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, saccharide or mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble oligomer (“O” in the conjugate formula O—X-D) can haveany of a number of different geometries. For example, “O” (in theformula O—X-D) can be linear, branched, or forked. Most typically, thewater-soluble oligomer is linear or is branched, for example, having onebranch point. Although much of the discussion herein is focused uponpoly(ethylene oxide) as an illustrative oligomer, the discussion andstructures presented herein can be readily extended to encompass any ofthe water-soluble oligomers described above.

The molecular weight of the water-soluble oligomer, excluding the linkerportion, is generally relatively low. Exemplary values of the molecularweight of the water-soluble polymer include, in some embodiments: belowabout 5000; below about 4500; below about 4000; below about 3500; belowabout 3000; below about 2500; below about 2000; below about 1500; belowabout 1400; below about 1300; below about 1200; below about 1100; belowabout 1000; below about 900; below about 800; below about 700; belowabout 600; below about 500; below about 400; below about 300; belowabout 200; and below about 100 Daltons.

Exemplary ranges of molecular weights of the water-soluble oligomer(excluding the linker) include, in some embodiments: from about 100 toabout 1400 Daltons; from about 100 to about 1200 Daltons; from about 100to about 800 Daltons; from about 100 to about 500 Daltons; from about100 to about 400 Daltons; from about 200 to about 500 Daltons; fromabout 200 to about 400 Daltons; from about 75 to 1000 Daltons; and fromabout 75 to about 750 Daltons.

Preferably, the number of monomers in the water-soluble oligomer fallswithin one or more of the following ranges: between about 1 and about 30(inclusive); between about 1 and about 25; between about 1 and about 20;between about 1 and about 15; between about 1 and about 12; betweenabout 1 and about 10. In certain instances, the number of monomers inseries in the oligomer (and the corresponding conjugate) is one of 1, 2,3, 4, 5, 6, 7, or 8. In additional embodiments, the oligomer (and thecorresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 monomers in series. In yet further embodiments, the oligomer(and the corresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 monomers in series.

When the water-soluble oligomer has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10monomers, these values correspond to a methoxy end-capped oligo(ethyleneoxide) having a molecular weights of 75, 119, 163, 207, 251, 295, 339,383, 427, and 471 Daltons, respectively. When the oligomer has 11, 12,13, 14, or 15 monomers, these values correspond to methoxy end-cappedoligo(ethylene oxide) having molecular weights corresponding to 515,559, 603, 647, and 691 Daltons, respectively.

In those instances where a bimodal oligomer is employed, the oligomerwill possess a bimodal distribution centering around any two of theabove numbers of monomers. Ideally, the polydispersity index of eachpeak in the bimodal distribution, Mw/Mn, is 1.01 or less, and even morepreferably, is 1.001 or less, and even more preferably is 1.0005 orless. Most preferably, each peak possesses a MW/Mn value of 1.0000. Forinstance, a bimodal oligomer may have any one of the following exemplarycombinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and soforth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7,4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth;6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and8-9, 8-10, and so forth.

In addition, the oligomer of the invention can be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, etc.) can beprepared by mixing purified monodisperse oligomers to obtain a desiredprofile of oligomers (a mixture of two oligomers differing only in thenumber of monomers is bimodal; a mixture of three oligomers differingonly in the number of monomers is trimodal; a mixture of four oligomersdiffering only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range. Ascan be seen from FIG. 10, commercially available PEGs are typicallypolydisperse mixtures, even for low molecular weight materials. Themethoxy-PEG sample shown was analyzed by mass spectrometry, and althoughlabeled as methoxy-PEG-350, the reagent was found to contain 9 differentPEG oligomer components, each differing in the number of monomersubunits. For the purposes of the present invention, that is to say, toprepare conjugates having the features described herein, polydispersepolymers are not particularly preferred, since small changes in thenumber of monomers have been discovered to have a profound effect on theproperties of the resulting conjugates. Such effects would likely bedampened or even undetectable in a conjugate mixture prepared using apolydisperse oligomer. Moreover, commercial batches of polydispersepolymers (or oligomers) are often highly variable in their composition,and for this reason, are not particularly preferred for the presentapplication, where batch-to-batch uniformity is a desirable feature foran oligomer as described herein.

As described above, the water-soluble oligomer is obtained from acomposition that is preferably unimolecular or monodisperse. That is,the oligomers in the composition possess the same discrete molecularweight value rather than a distribution of molecular weights. Somemonodisperse oligomers can be purchased from commercial sources such asthose available from Sigma-Aldrich, or alternatively, can be prepareddirectly from commercially available starting materials such asSigma-Aldrich. For example, oligoethylene glycols of the invention canbe prepared as described, e.g., in Chen Y., Baker, G. L., J. Org. Chem.,6870-6873 (1999), or in WO 02/098949 A1. Alternatively, such oligomerscan be prepared as described herein in Example 9.

As described above, one aspect of the invention is an improved method ofpreparing a monodisperse oligomers such as an oligo(ethylene oxide).These oligomers can be used in any of a variety of applications,including but not limited to preparing a small moleculedrug-water-soluble oligomer conjugate having the beneficial propertiesset forth above.

In order to provide the desired monodisperse oligomers, a new approachwas used. It was discovered that halo-terminated oligomer reagents aremore reactive and produce higher yields of monofunctional products incomparison to previously described reagents.

Thus, the present invention also includes a method for preparingmonodisperse oligomer compositions. The method involves reacting ahalo-terminated oligomer such as an oligo(ethylene oxide) having (m)monomers with a hydroxyl-terminated oligo(ethylene oxide) having (n)monomers. Generally, the halo group on the halo terminated oligoethyleneglycol is a chloro, bromo or iodo group. Preferably, however, the halogroup is bromo. The reaction is carried out under conditions effectiveto displace the halo group from the halo-terminated oligomer to therebyform an oligo(ethylene oxide) having (m)+(n) monomer subunits(OEG_(m+n)), where (m) and (n) each independently range from 1-10. Thatis to say, each of (m) and (n) is independently 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. Preferably, (m) and (n) each independently range from 1 toabout 6. In selected embodiments, (m) is 1, 2, or 3 and (n) ranges from1-6. In other instances, (m) is 1, 2, or 3, and (n) ranges from 2-6.Typically, the reaction is carried out in the presence of a strong baseeffective to convert the hydroxyl group of the hydroxyl-terminatedoligoethylene oxide into the corresponding alkoxide species. Suitablebases include sodium, potassium, sodium hydride, potassium hydride,sodium methoxide, potassium methoxide, sodium tert-butoxide, andpotassium tert-butoxide. In a preferred embodiment, the halo-terminatedoligoethylene glycol possesses an end-capping group such as methoxy orethoxy.

Representative hydroxy-terminated oligo(ethylene glycol)s correspond tothe structure HO—(CH₂CH₂O)_(n)—H, where (n) is as described above. Themethod then preferably includes the step of converting the terminalhydroxyl group of OEG_(m+n) into a halo group, —X, to form OEG_(m++n)-X.The above steps are then repeated until a unimolecular oligomer havingthe desired number of subunits is obtained.

An illustrative reaction scheme is as follows.

As shown, the method involves the coupling of two unimolecular oligomerspecies by employing a substitution reaction where a halide on oneoligomer, preferably an oligomeric ethylene oxide, and even morepreferably, a halo-derivatized oligoethylene oxide methyl ether, isreacted with an oligoethylene glycol-alkoxide to generate thecorresponding oligomer (see reaction 1 above).

The alkoxide is typically generated from the corresponding oligoethyleneoxide by converting the terminal hydroxyl to the corresponding alkoxidein the presence of a strong base. The reaction is generally carried outin an organic solvent such as tetrahydrofuran (“THF”) at temperaturesranging from about 0° C. to about 80° C. Reaction times typically rangefrom about 10 minutes to about 48 hours. The resultant product, in theexemplary reaction above, an end-capped oligoethylene oxide, contains asum of the number of monomers of the halo-derivatized oligomer and thenumber of monomers in the oligoethylene glycol alkoxide [(m)+(n)].Yields typically range from about 25% to about 75% for the purifiedcoupled product, with yields most typically ranging from about 30 toabout 60%.

In the above example, the hydroxyl terminus in the product from reaction1 is then activated, if necessary, for coupling to a small molecule.Alternatively, if desired, the hydroxyl terminus in the exemplaryproduct shown above [in the above example having (m)+(n) subunits), isthen converted to a halide, preferably a bromide. Conversion of analcohol to an alkyl halide can by effected directly, or through anintermediate such as a sulfonate or haloformate. Conditions and reagentssuitable for effecting this transformation are found, for example, inLarock, R., “Comprehensive Organic Transformations”, VCH, 1994, pages353 to 363.

One preferred method is that set forth in Example 11. The stepwiseaddition of the oligoethylene oxide halide to an oligoethylene oxide isthen repeated as described above, to form an oligoethylene oxide having(m)+2(n) monomers, and so-forth. In this manner, discrete oligoethyleneoxide subunits are then added in a controlled, stepwise fashion to theexisting monomeric (unimolecular) oligomeric ethylene oxide product, toensure preparation of a well-defined oligomer having an exact number ofsubunits.

Commonly available are unimolecular oligoethylene glycols having fromabout 1-3 monomer subunits (Sigma-Aldrich). Use of a halo-substitutedoligomeric ethylene glycol reactant represents an improvement overexisting methods, e.g., employing the mesylate, since the approachprovided herein results in improved yields, shorter reaction times andmilder reaction conditions due to the higher reactivity of the halide,and in particular, the bromo-substituted oligoethylene glycol reagent.Oligomers thus prepared are typically purified prior to further use, forexample, by one or more of the following methods: chromatography such asHPLC, ion exchange chromatography, column chromatography, precipitation,or recrystallization. Purity is then confirmed by any of a number ofanalytical techniques, such as NMR, GPC, and FTIR. Products thus formedare then suitable for further use.

The linker or linkage of the invention may be a single atom, such as anoxygen or a sulfur, two atoms, or a number of atoms. A linker istypically but is not necessarily linear in nature. The linkage, “X” (inthe O—X-D formula), is hydrolytically stable, and is preferably alsoenzymatically stable. Preferably, the linkage “X” is one having a chainlength of less than about 12 atoms, and preferably less than about 10atoms, and even more preferably less than about 8 atoms or even morepreferably less than about 5 atoms, whereby length is meant the numberof atoms in a single chain, not counting substituents. For instance, aurea linkage such as this, R_(oligomer-NH—(C═O)—NH—R′) _(drug), isconsidered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selectedembodiments, the linkage does not comprise further spacer groups. Smalllinkages are preferred and lend themselves to the nature of the presentinvention, since small linkages such as these are less likely todominate or overshadow the effect of an addition of one or a smallnumber of monomer subunits on the difference in transport properties ofthe conjugates of the invention.

In some instances, the linker “X” is hydrolytically stable and comprisesan ether, amide, urethane, amine, thioether, urea, or a carbon-carbonbond. Functional groups such as those discussed below, and illustratedin the working examples, are typically used for forming the linkages.The linkage may less preferably also comprise (or be adjacent to orflanked by) spacer groups, as described further below. Spacers are mostuseful in instances where the bioactivity of the conjugate issignificantly reduced due to the positioning of the oligomer on theparent drug.

More specifically, in selected embodiments, a linker of the invention,L, may be any of the following: —O—, —NH—, —S—, —C(O)—, C(O)—NH,NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,CH₂—CH₂—O—S—O—CH₂—CH₂—CH₂—, CH₂—O—CH₂—CH₂—, CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—S—O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,CH₂—CH₂—CH₂—CH₂—O—S—C(O)—NH—CH₂—, C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—,CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—,CH₂—CH₂—C(O)—NH—CH₂—, CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—,CH₂—CH₂—NH—C(O)—CH₂—, NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂,—CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—,—CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a series of atoms is notconsidered as a linkage when the series of atoms is immediately adjacentto an oligomer segment, and the series of atoms is but another monomersuch that the proposed linkage would represent a mere extension of theoligomer chain.

The linkage “X” between the oligomer and the small molecule is typicallyformed by reaction of a functional group on a terminus of the oligomerwith a corresponding functional group within the small molecule drug.Illustrative reactions are described briefly below. For example, anamino group on an oligomer, “O,” may be reacted with a carboxylic acidor an activated carboxylic acid derivative on the small molecule, orvice versa, to produce an amide linkage. Alternatively, reaction of anamine on an oligomer with an activated carbonate (e.g. succinimidyl orbenzotriazyl carbonate) on the drug, or vice versa, forms a carbamatelinkage. Reaction of an amine on an oligomer with an isocyanate(R—N═C═O) on a drug, or vice versa, forms a urea linkage(R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group onan oligomer with an alkyl halide, or halide group within a drug, or viceversa, forms an ether linkage. In yet another coupling approach, a smallmolecule having an aldehyde function is coupled to an oligomer aminogroup by reductive amination, resulting in formation of a secondaryamine linkage between the oligomer and the small molecule.

A particularly preferred oligomer is an oligomer bearing an aldehydefunctional group. In this regard, the oligomer will have the followingstructure: CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and7. Preferred (n) values include 3, 5, and 7, and preferred (p) valuesinclude 2, 3, and 4. In addition, the carbon atom alpha to the —C(O)Hmoiety can optionally be substituted with alkyl. The oligomer reagent ispreferably provided as a monodisperse composition.

Typically, the terminus of the oligomer not bearing a functional groupis capped to render it unreactive. When the oligomer does includes afurther functional group at a terminus other than that intended forformation of a conjugate, that group is either selected such that it isunreactive under the conditions of formation of the linkage “X,” or itis protected during the formation of the linkage “X.”

As stated above, the oligomer includes a functional group for forming asmall molecule conjugate having the properties described herein. Thefunctional group typically comprises an electrophilic or nucleophilicgroup for covalent attachment to a small molecule, depending upon thereactive group contained within or introduced into the small molecule.Examples of nucleophilic groups that may be present in either theoligomer or the small molecule include hydroxyl, amine, hydrazine(—NHNH₂), hydrazide (—C(O)NHNH₂), and thiol. Preferred nucleophilesinclude amine, hydrazine, hydrazide, and thiol, particularly amine. Mostsmall molecule drugs for covalent attachment to an oligomer will possessa free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, etc., as well as hydrates orprotected derivatives of any of the above moieties (e.g. aldehydehydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal). Another useful conjugation reagent is2-thiazolidine thione.

As noted above, an “activated derivative” of a carboxylic acid refers toa carboxylic acid derivative which reacts readily with nucleophiles,generally much more readily than the underivatized carboxylic acid.Activated carboxylic acids include, for example, acid halides (such asacid chlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g. hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable. Asmentioned above, most preferred are conjugates having a hydrolyticallystable linkage between the oligomer and the drug. Carbonates, includingsuccinimidyl, imidazolyl, and benzotriazole carbonates, react with aminogroups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl oramino groups to form, respectively, carbamate (RNH—C(O)—OR′) or urea(RNH—C(O)—NHR′) linkages. Aldehydes, ketones, glyoxals, diones and theirhydrates or alcohol adducts (i.e. aldehyde hydrate, hemiacetal, acetal,ketone hydrate, hemiketal, and ketal) are preferably reacted withamines, followed by reduction of the resulting imine, if desired, toprovide an amine linkage (reductive amination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups which can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

For instance, the preparation of an exemplary oligomeric conjugate ofretinoic acid is described in detail in Example 1. Briefly, the smallmolecule, retinoic acid, which contains a reactive carboxyl group, iscoupled to an amino-activated oligomeric ethylene glycol, to provide aconjugate having an amide group covalently linking the small molecule tothe oligomer. The covalent attachment of each a PEG 3-mer (meaning anoligomeric ethylene glycol having 3 ethylene glycol monomer subunits), aPEG 7-mer, and a PEG 11-mer to retinoic acid is described.

Further, the preparation of an oligomer-conjugate of naloxone isdescribed in Example 4. In this representative synthesis, followingprotection of an aromatic hydroxyl group, a keto group in naloxone isreduced to the corresponding hydroxyl, which is then coupled to anoligomeric ethylene glycol halide to result in an ether (—O—) linkedsmall molecule conjugate. Interestingly, in this example, reduction ofthe hydroxyl group in naloxone resulted in formation of twostereoisomers differing in the orientation of the hydroxyl group. Thecorresponding oligomeric conjugates were prepared and separated, andshown to have somewhat different characteristics, to be discussed ingreater detail below. This represents another feature of the invention,that is, the preparation/isolation of single isomers of oligomer-smallmolecule conjugates, and uses thereof.

The conjugates of the invention exhibit a reduced biological barriercrossing rate as previously described. Moreover, the conjugatestypically maintain at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,or more of the bioactivity of the unmodified parent small molecule drug.For a given small molecule drug having more than one reactive sitesuitable for modification, it may be necessary to carry out molecularmodeling, or in vivo or in vitro biological activity assays to assessthe biological activity of the resulting conjugate and determine thesite most suitable for covalent attachment of an oligomer. See forexample the illustrative bioactivity data in Table VI for variousoligomer conjugates of naloxone and derivatized naloxone, 6-NH₂-naloxoneand 6-OH-naloxol. In this investigation, variables included the site ofchemical modification on the parent drug, type of covalent linkage,stereochemistry, and size of oligomer covalently attached to the drugmoiety. As can be seen from the data, the bioactivities of theconjugates ranged from about 5% to about 35% of the bioactivity of theparent drug.

It has been discovered that stable covalent attachment of small,water-soluble oligomers to orally bioavailable small molecule drugs iseffective to significantly alter the properties of these molecules,thereby making them more clinically effective. More specifically,covalent attachment of monodisperse oligomers such as oligoethyleneoxide is often effective to reduce, or in some cases, eliminate, adrug's transport across the blood brain barrier, which then translatesinto a significant reduction in central nervous system-related sideeffects. The selection of an optimally sized oligomer can be conductedas follows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to a small molecule drug. Preferably, the drug isorally bioavailable, and on its own, exhibits a biological membranecrossing rate. Next, the ability of the conjugate to cross thebiological membrane is determined using an appropriate model andcompared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. A beneficial conjugate in accordance with the invention isbioactive, since the linkage is hydrolytically stable and does notresult in release of unmodified drug upon administration. Thus, the drugin conjugated form should be bioactive, and preferably, maintains asignificant degree of bioactivity relative to the parent drug, i.e.,greater than about 30% of the bioactivity of the parent drug, or evenmore preferably, greater than about 50% of the bioactivity of the parentdrug. Then, the above steps are repeated using oligomers of the samemonomer type but having a different number of subunits.

Because the gastro-intestinal tract (“GIT”) limits the transport of foodand drugs from the digestive lumen in to blood and the lymph, the GITrepresents another barrier for which the conjugate may be tested. TheGIT barrier, however, represents a barrier that must not block theconjugates when the conjugate is intended for oral administration forsystemic delivery. The GIT barrier consists of continuous layers ofintestinal cells joined by tight junctions in the intestinal epithelia.

For each conjugate whose ability to cross a biological membrane isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results, that isto say, based upon the sequential addition of increasing numbers ofdiscrete monomers to a given small molecule at a given position orlocation within the small molecule, it is possible to determine the sizeof the oligomer most effective in providing a conjugate having anoptimal balance between reduction in biological membrane crossing, oralbioavailability, and bioactivity. The small size of the oligomers makessuch screenings feasible, and allows one to effectively tailor theproperties of the resulting conjugate. By making small, incrementalchanges in oligomer size, and utilizing an experimental design approach,one can effectively identify a conjugate having a favorable balance ofreduction in biological membrane crossing rate, bioactivity, and oralbioavailability. In some instances, attachment of an oligomer asdescribed herein is effective to actually increase oral bioavailabilityof the drug.

In view of the present disclosure, one of ordinary skill in the art,using routine experimentation, can determine a best-suited molecularsize and linkage for improving oral bioavailability by first preparing aseries of oligomers with different weights and functional groups andthen obtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumarate, and combinationsthereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th), Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, and the like. Binders are used to impart cohesive qualities to atablet, and thus ensure that the tablet remains intact. Suitable bindermaterials include, but are not limited to, starch (including corn starchand pregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, waxes, and natural andsynthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, microcrystalline cellulose, ethylcellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricantsare used to facilitate tablet manufacture, promoting powder flow andpreventing particle capping (i.e., particle breakage) when pressure isrelieved. Useful lubricants are magnesium stearate, calcium stearate,and stearic acid. Disintegrants are used to facilitate disintegration ofthe tablet, and are generally starches, clays, celluloses, algins, gums,or crosslinked polymers. Fillers include, for example, materials such assilicon dioxide, titanium dioxide, alumina, talc, kaolin, powderedcellulose, and microcrystalline cellulose, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form(typically as a lyophilizate or precipitate, which can be in the form ofa powder or cake), as well as formulations prepared for injection, whichare typically liquid and requires the step of reconstituting the dryform of parenteral formulation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions, eachtypically being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The pulmonary formulations may take various forms. Examples ofpharmaceutically acceptable excipients for pulmonary delivery include,but are not limited to, lipids, metal ions, surfactants, amino acids,peptides, carbohydrates, buffers, salts, polymers, and the like, andcombinations thereof. Vehicles for pulmonary delivery and processes ofmaking such vehicles are disclosed, e.g., in U.S. Pat. Nos. 6,518,239and 6,565,885, which are incorporated herein by reference.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection.

In instances where parenteral administration is utilized, it may benecessary or desirable to employ somewhat larger oligomers than thosedescribed previously, with molecular weights ranging from about 500 to30K Daltons (e.g., having molecular weights of about 500, 1000, 2000,2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that a reduction in first pass metabolism may be achieved relative tothe parent drug. See for example the supporting results in Example 8.Such a result is advantageous for many orally administered drugs thatare substantially metabolized by passage through the gut. In this way,clearance of the conjugate can be modulated by selecting the oligomermolecular size, linkage, and position of covalent attachment providingthe desired clearance properties. One of ordinary skill in the art candetermine the ideal molecular size of the oligomer based upon theteachings herein. All articles, books, patents, patent publications andother publications referenced herein are incorporated by reference intheir entireties. Preferred reductions in first pass metabolism for aconjugate as compared to the corresponding nonconjugated small drugmolecule include: at least about 10%, at least about 20%, at least about30; at least about 40; at least about 50%; at least about 60%, at leastabout 70%, at least about 80% and at least about 90%.

Thus, the invention provides a method for reducing the metabolism of anactive agent. The method comprises: providing monodisperse or bimodalconjugates, each conjugate comprised of a moiety derived from a smallmolecule drug covalently attached by a stable linkage to a water-solubleoligomer, wherein said conjugate exhibits a reduced rate of metabolismas compared to the rate of metabolism of the small molecule drug notattached to the water-soluble oligomer; and administering the conjugateto a patient. Typically, administration is carried out via one type ofadministration selected from the group consisting of oraladministration, transdermal administration, buccal administration,transmucosal administration, vaginal administration, rectaladministration, parenteral administration, and pulmonary administration.

Although useful in reducing many types of metabolism (including bothPhase I and Phase II metabolism), the conjugates are particularly usefulwhen the small molecule drug is metabolized by a hepatic enzyme (e.g.,one or more of the cytochrome P450 isoforms) and/or by one or moreintestinal enzymes.

In accordance with some embodiments of the present invention, methods ofmodifying the route of absorption of a compound administered to asubject by a pulmonary route are provided. The methods comprisecovalently attaching a hydrophilic polymer to a compound to form acompound-polymer conjugate. In one example, the compound-polymerconjugate has a net hydrophilic character.

The conjugates exhibiting modified routes of absorption of a compoundcan comprise the structure: P-L-D, wherein P comprises a hydrophilicpolymer, L comprises a linker, and D comprises a drug. In accordancewith embodiments of the invention, the polymer P can be chosen from anyof the water soluble oligomers O that exhibit hydrophilic characterdescribed herein. In one embodiment, the polymer P can be chosen from amonodisperse or bimodal population of oligomers as described herein withrespect with to the water soluble oligomers O. In other embodiments, thepolymers can be chosen from a polydisperse population of polymerscomprising hydrophilic oligomers O of varying molecular weights. In oneembodiment, the polymer P can be a polydisperse population ofpolyethylene glycols (PEGs). In another embodiment, the PEG can be amonodisperse or bimodal PEG population. The PEG can have any suitablegeometry. For example, the PEG can be a linear PEG, branched PEG, forkedPEG, and a dumbbell PEG, and combinations thereof.

In instances in which pulmonary administration is utilized, themolecular weight may be less than about 5000 Daltons, or less than about4000 Daltons, or less than about 3000 Daltons, or less than about 2000Daltons, or less than about 1000 Daltons. In some embodiments, theranges of molecular weights for pulmonary administration will range fromabout 50 Daltons to about 3500 Daltons. However, the molecular weightsof the polymers are not so limited. The polymer P can have a weightaverage molecular weight of from about x to about y, wherein x and yinclude, but are not limited to: 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900,2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500,3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4050, 4100, 4150,4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750,4800, 4850, 4900, 4950, 5000, 5000, 5050, 5100, 5150, 5200, 5250, 5300,5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900,5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500,6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100,7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700,7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300,8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900,8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500,9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050,10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550,10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050,11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550,11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050,12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550,12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, 13000, 13050,13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550,13600, 13650, 13700, 13750, 13800, 13850, 13900, 14000, 14050, 14100,14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600,14650, 14700, 14750, 14800, 14850, 14900, 14950, 15000, 15000, 15050,15100, 15150, 15200, 15250, 15300, 15350, 15400, 15450, 15500, 15550,15600, 15650, 15700, 15750, 15800, 15850, 15900, 15950, 16000, 16050,16100, 16150, 16200, 16250, 16300, 16350, 16400, 16450, 16500, 16550,16600, 16650, 16700, 16750, 16800, 16850, 16900, 16950, 17000, 17050,17100, 17150, 17200, 17250, 17300, 17350, 17400, 17450, 17500, 17550,17600, 17650, 17700, 17750, 17800, 17850, 17900, 17950, 18000, 18050,18100, 18150, 18200, 18250, 18300, 18350, 18400, 18450, 18500, 18550,18600, 18650, 18700, 18750, 18800, 18850, 18900, 18950, 19000, 19050,19100, 19150, 19200, 19250, 19300, 19350, 19400, 19450, 19500, 19550,19600, 19650, 19700, 19750, 19800, 19850, 19900, 19950, and 20000Daltons. Thus, in one example, the weight average molecular weight ofthe polymer P is between about 50 and about 20000 Daltons. In anotherexample, the weight average molecular weight of the polymer P is betweenabout 50 and about 5000 Daltons, or between about 1000 and about 3500Daltons. In yet another example, the weight average molecular weight ofthe polymer is between about 50 and about 1000 Daltons.

The linker L may be a hydrolytically stable linker X as described above.In other embodiments, the linker L may be linker that is unstable underphysiological conditions or lung conditions such that the drug D can bereleased from the drug-polymer conjugate into the lung. For example, thelinker X can comprise hydrolytically unstable linkers including, but notlimited to, ester, thioester, and amides. It will be understood that thelinker L may be chosen to be stable or unstable as desired for aparticular application. The drug D may be as described above. In oneexample, the drug D can comprise a drug having a molecular weight ofless than about 1500 or less than about 1000. It will be understood thatthe drug D can be a drug in its active form or a prodrug. It will befurther understood that the drug D will be a drug in which pulmonaryadministration is desirable.

In accordance with further embodiments of the present invention, methodsof controlling the rate of systemic absorption of a drug (also referredto herein as “small molecule”) pulmonarily administered are provided.The methods comprise covalently attaching to the drug a hydrophilicpolymer molecule to form a drug-polymer conjugate. As noted above, theattachment of the hydrophilic polymer can have the effect of increasingthe ability of an otherwise non-absorbed drug to be absorbed. Further,the rate of systemic absorption of the drug can be decreased byincreasing the size of the attached hydrophilic polymer. Without wishingto be bound by theory, it is believed that increasing the size of thehydrophilic polymer slows the crossing of the conjugate through thepulmonary epithelium.

In some embodiments, the half-life of elimination of the drug-conjugatefrom the lung will be from about 0.5 hours to about 12 hours, or fromabout 1 to about 10 hours, or from about 1.5 to about 8 hours, or fromabout 2 to about 6 hours, or from about 2.5 to about 4 hours. In someembodiments, the half-life of elimination is less than about 12, 11.5,11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05,0.04, 0.03, 0.02, or 0.01 hours. In one or more embodiments, the rate ofcrossing, K, is less than about 0.057, 0.060, 0.063, 0.066, 0.069,0.073, 0.077, 0.082, 0.087, 0.092, 0.099, 0.107, 0.117, 0.126, 0.139,0.154, 0.173, 0.462, 0.693, or 1.39 per hour. Generally, the half-lifeof elimination from the lung of pulmonary administered drug-polymerconjugates according to the invention can be predicted by the followingequation: t_(1/2)-el=12.84*(1-e^(−kMW)), where k=0.000357, MW=molecularweight in Daltons, and t_(1/2)=elimination half-life in hours.

In some embodiments, the half-life of elimination from the lung of theunconjugated drug is less than about 180, 170, 160, 150, 140, 130, 120,110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 minute. In someembodiments, the drug-polymer conjugate according to the invention, hasa half-life of elimination from the lung that is 1.5-fold or moregreater than the half-life of elimination from the lung of theunconjugated drug. For example, the half-life of elimination from thelung may be increased by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0,10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or even1000-fold.

In some embodiments, the attachment of the hydrophilic polymer can havethe effect of causing the drug-polymer conjugate to have a lower log Pthan the parent unconjugated drug (as measured in the shake-flaskmethod). In some embodiments, the log P of the drug-polymer conjugatewill be lower than about 2.5, 2.0, 1.5, 1.0, 0.5, 0, −0.1, −0.5, −1.0,−1.5, −2.0, −2.5, −3.0, −3.5, or −4.0. The difference in log P betweenthe conjugated drug and the unconjugated drug may range from about 0.2to about 1.5, such as about 0.5 to about 1.2, about 0.4 to about 1, orabout 0.5 to about 0.8.

The drug-polymer conjugate may be as described above in reference to theconjugate having a structure of P-L-D. In one example, the hydrophilicpolymer is a polydisperse PEG. In one example, the hydrophilic polymerhas a weight average molecular weight of between about 50 to about 4000.In another example, the hydrophilic polymer has a weight averagemolecular weight of between about 1000 to about 3500 or about 50 toabout 1350. In yet another example, the drug-polymer conjugate exhibitsa net hydrophilic character.

In accordance with further embodiments of the present invention, methodsof controlling the lung residence time of a drug pulmonarilyadministered are provided. The methods comprise covalently attaching tothe drug a hydrophilic polymer molecule to form a drug-polymer conjugateand delivering the drug-polymer conjugate by pulmonary administration.The drug-polymer conjugate may be as described above in reference to theconjugate having a structure of P-L-D. In one example, the hydrophilicpolymer is a polydisperse PEG. In one example, the hydrophilic polymerhas a weight average molecular weight of between about 50 and about 4000Daltons. In another example, the hydrophilic polymer has a weightaverage molecular weight of between about 1000 and about 3500 Daltons orabout 50 and about 1350 Daltons. In yet another example, thedrug-polymer conjugate exhibits a net hydrophilic character. In someembodiments, by attaching the polymer to the drug, the residence time ofthe drug in the lung is increased, as compared to the residence time ofa free form of the drug.

In accordance with some embodiments of the present invention,pharmaceutical compounds for pulmonary administration are provided. Thepharmaceutical compound comprises a drug covalently attached to ahydrophilic polymer. The pharmaceutical compound may have a nethydrophilic character. The pharmaceutical compound can comprise thedrug-polymer conjugates P-L-D described herein. In one example, theweight average molecular weight of the pharmaceutical compound is fromabout 50 to about 3500 Daltons.

In some embodiments, a composition comprising the pharmaceuticalcompound and at least one pharmaceutically acceptable excipient isprovided. The at least one of the pharmaceutically acceptable excipientscan be selected from the pharmaceutically acceptable excipientsdescribed herein. It will be understood that the composition can beprovided in any form suitable for pulmonary administration. For example,the composition can be provided in liquid or dry form, including drypowder form. In one example, the composition can be provided as anaerosol. In one embodiment, the composition can be a spray-driedcomposition. In one example, the composition can comprise particleshaving a mass median aerodynamic diameter (MMAD) of less than about 10microns or less than about 5 microns. The composition having an MMAD ofless than about 10 microns or less than about 5 microns can comprise anaerosolized composition. Additionally, the composition can be providedin any suitable delivery device. For example, the composition can beprovided in an inhaler device.

The composition may be used to treat any condition that can be remediedor prevented by administration of the particular pharmaceuticalcompound. Those of ordinary skill in the art appreciate which conditionsa specific pharmaceutical compound can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature.

The unit dosage of any given pharmaceutical composition be administeredin a variety of dosing schedules depending on the judgment of theclinician, needs of the patient, and so forth. The specific dosingschedule will be known by those of ordinary skill in the art or can bedetermined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Once the clinical endpoint has beenachieved, dosing of the composition can be halted. In one embodiment,the drug-polymer conjugate can be provided in a unit dosage form. Forexample, the drug-polymer conjugate can be provided in a unit dosageform that is delivered by an inhaler device.

EXAMPLES

It is to be understood that while the invention has been described inconjunction with certain preferred and/or specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages, and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofillustrative unimolecular PEG-mers is described in Example 9. Alloligo(ethylene glycol) methyl ethers employed in the Examples below weremonodisperse and chromatographically pure, as determined by reversephase chromatography.

All ¹H NMR (nuclear magnetic resonance) data was generated by a 300 MHzNMR spectrometer manufactured by Bruker. A list of certain compounds aswell as the source of the compounds is provided below.

2-Bromoethyl methyl ether, 92%, Aldrich;

1-Bromo-2-(2-methoxyethoxy)ethane, 90%, Aldrich;

CH₃(OCH₂CH₂)₃Br was prepared from CH₃(OCH₂CH₂)₃OH;

Tri(ethylene glycol) monomethyl ether, 95%, Aldrich;

Di(ethylene glycol), 99%, Aldrich;

Tri(ethylene glycol), 99%, Aldrich;

Tetra(ethylene glycol), 99%, Aldrich;

Penta(ethylene glycol), 98%, Aldrich;

Hexa(ethylene glycol), 97%, Aldrich;

Sodium hydride, 95% dry powder, Aldrich;

Methansulfonyl chloride, 99%, ACE;

Tetrabutyl ammonium bromide, Sigma

Example 1 Synthesis of CH₃(OCH₂CH₂)₃—NH-13-Cis-RetentamidePEG₃-13-cis-RA

PEG₃-13-cis-RA was prepared. The overview of the synthesis is providedbelow.

0.1085 grams of CH₃(OCH₂CH₂)₃—NH₂ (0.6656 mmoles), 0.044 grams of1-hydroxybenzyltriazole (“HOBT,” 0.3328 mmoles), and 0.200 g of13-cis-retinoic acid (“13-cis-RA,” 0.6656 mmoles) were dissolved in 10mL of benzene. To this solution was added 0.192 grams of1,3-dicyclohexylcarbodiimide (“DCC,” 0.9318 mmoles) and the reactionmixture was stirred overnight at room temperature. The reaction mixturewas filtered and the solvent was removed using rotary evaporation. Thecrude product was further dried under vacuum, dissolved in 20 mL ofdichloromethane, and the organic phase was washed twice with 15 mL ofdeionized water. The organic phase was dried over Na₂SO₄, filtered, andthe solvent removed by rotary evaporation. To the recovered product wasadded 2 drops of dichloromethane containing 50 ppm butylatedhydroxytoluene and the product was dried under vacuum. Yield 0.335 g. ¹HNMR (DMSO): δ 1.02 (singlet, 2 CH₃), 1.67 (singlet, CH₃), 3.5 (broadmultiplet, PEG), 6.20 (m, 3H).

Example 2 Synthesis of CH₃—(OCH₂CH₂)₇—NH-13-cis-Retinamide(PEG₇-13-cis-RA

0.2257 grams of CH₃(OCH₂CH₂)₇—NH₂ (0.6656 mmoles), 0.044 grams of1-hydroxybenzyltriazole (0.3328 mmoles), and 0.200 grams of13-cis-retinoic acid (0.6656 mmoles) were dissolved in 10 mL of benzene.To this solution was added 0.192 g 1,3-dicyclohexylcarbodiimide (0.9318mmoles) and the resulting reaction mixture was stirred overnight at roomtemperature. The reaction mixture was filtered, the solvent removedusing rotary evaporation, and the product dried under vacuum. Theproduct was dissolved in 20 mL dichloromethane and the solution waswashed twice with 15 mL deionized water. The organic phase was driedover Na₂SO₄, filtered, and the solvent removed using rotary evaporation.To the recovered product was added 2 drops of dichloromethane containing50 ppm butylated hydroxytoluene, and the product was dried under vacuum.Yield 0.426 g. ¹H NMR (DMSO): δ 1.01 (s, 2 CH₃), 1.68 (s, CH₃), 3.5 (brm, PEG), 6.20 (m, 3H).

CH₃—(OCH₂CH₂)₅—NH-13-cis-retinamide (“PEG₅-13-cis-RA”) was similarlyprepared using this procedure except that CH₃(OCH₂CH₂)₅—NH₂(“mPEG₅-NH₂”) was used in place of CH₃(OCH₂CH₂)₇—NH₂.

Example 3 Synthesis of CH₃—OCH₂CH₂)₁₁—NH-13-Cis-RetinamidePEG₁₁-13-cis-RA

0.349 grams of CH₃(OCH₂CH₂)₁₁—NH₂ (0.6789 mmoles), 0.044 grams of1-hydroxybenzyltriazole (0.3328 mmoles), and 0.204 grams of13-cis-retinoic acid (0.6789 mmoles) was dissolved in 10 mL of benzene.To this solution was added 0.192 g 1,3-dicyclohexylcarbodiimide (0.9318mmoles) and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was filtered and the solvent distilledoff using rotary evaporation. The product was dried under vacuum anddissolved in 20 mL dichloromethane. The solution was washed twice with15 mL of deionized water and the organic phase dried over Na₂SO₄. Thesolution was filtered and the solvent was distilled off by rotaryevaporation. To the recovered product was added 2 drops ofdichloromethane containing 50 ppm butylated hydroxytoluene, and theproduct was dried under vacuum. Yield 0.541 g. ¹H NMR (DMSO): δ 1.01 (s,2 CH₃), 1.68 (s, CH₃), 3.5 (br m, PEG), 6.20 (m, 3H).

Example 4 Synthesis of PEG₃-3-Naloxol

The structure of the naloxol, an exemplary small molecule drug, is shownbelow.

This molecule was prepared (having a protected hydroxyl group) as partof a larger synthetic scheme as described in Example 5.

Example 5 Synthesis of α,β-6-CH₃—(OCH₂CH₂)₁-Naloxol (α,β-PEG₁-Nal)

α,β-PEG₁-naloxol was prepared. The overview of the synthesis is providedbelow.

5.A. Synthesis of 3-MEM-naloxone

Diisopropylethylamine (390 mg, 3.0 mmole) was added to a solution ofnaloxone.HCl.2H₂O (200 mg, 0.50 mmole) in CH₂Cl₂ (10 mL) with stirring.Methoxyethyl chloride (“MEMCl,” 250 mg, 2.0 mmole) was then addeddropwise to the above solution. The solution was stirred at roomtemperature under N₂ overnight.

The crude product was analyzed by HPLC, which indicated that3-MEM-O-naloxone (1) was formed in 97% yield. Solvents were removed byrotary evaporation to yield a sticky oil.

5.B. Synthesis of α and β Epimer Mixture of 3-MEM-Naloxol (2)

3 mL of 0.2 N NaOH was added to a solution of 3-MEM-naloxone (1)(obtained from 5.A. above, and used without further purification) in 5mL of ethanol. To this was added a solution of NaBH₄ (76 mg, 2.0 mmole)in water (1 mL) dropwise. The resulting solution was stirred at roomtemperature for 5 hours. The ethanol was removed by rotary evaporationfollowed by addition of a solution of 0.1 N HCl solution to destroyexcess NaBH₄ and adjust the pH to a value of 1. The solution was washedwith CHCl₃ to remove excess methoxyethyl chloride and its derivatives(3×50 mL), followed by addition of K₂CO₃ to raise the pH of the solutionto 8.0. The product was then extracted with CHCl₃ (3×50 mL) and driedover Na₂SO₄. The solvent was removed by evaporation to yield a colorlesssticky solid (192 mg, 0.46 mmole, 92% isolated yield based onnaloxone.HCl.2H₂O).

HPLC indicated that the product was an α and β epimer mixture of3-MEM-naloxol (2).

5.C. Synthesis of α and β Epimer Mixture of6-CH₃—OCH₂CH₂—O-3-MEM-Naloxol (3a).

NaH (60% in mineral oil, 55 mg, 1.38 mmole) was added into a solution of6-hydroxyl-3-MEM-naloxol (2) (192 mg, 0.46 mmole) in dimethylformamide(“DMF,” 6 mL). The mixture was stirred at room temperature under N₂ for15 minutes, followed by addition of 2-bromoethyl methyl ether (320 mg,2.30 mmole) in DMF (1 mL). The solution was then stirred at roomtemperature under N₂ for 3 hours.

HPLC analysis revealed formation of a mixture of α- andβ-6-CH₃—OCH₂CH₂—O-3-MEM-naloxol (3) in about 88% yield. DMF was removedby a rotary evaporation to yield a sticky white solid. The product wasused for subsequent transformation without further purification.

5.D. Synthesis of α and β Epimer Mixture of 6-CH₃—OCH₂CH₂-Naloxol (4)

Crude α- and β-6-CH₃—OCH₂CH₂—O-3-MEM-naloxol (3) was dissolved in 5 mLof CH₂Cl₂ to form a cloudy solution, to which was added 5 mL oftrifluoroacetic acid (“TFA”). The resultant solution was stirred at roomtemperature for 4 hours. The reaction was determined to be completebased upon HPLC assay. CH₂Cl₂ was removed by a rotary evaporator,followed by addition of 10 mL of water. To this solution was addedsufficient K₂CO₃ to destroy excess TFA and to adjust the pH to 8. Thesolution was then extracted with CHCl₃ (3×50 mL), and the extracts werecombined and further extracted with 0.1 N HCl solution (3×50 mL). The pHof the recovered water phase was adjusted to a pH of 8 by addition ofK₂CO₃, followed by further extraction with CHCl₃ (3×50 mL). The combinedorganic layer was then dried with Na₂SO₄. The solvents were removed toyield a colorless sticky solid.

The solid was purified by passage two times through a silica gel column(2 cm×30 cm) using CHCl₃/CH₃OH (30:1) as the eluent to yield a stickysolid. The purified product was determined by ¹H NMR to be a mixture ofα- and β epimers of 6-CH₃—OCH₂CH₂-naloxol (4) containing ca. 30% αepimer and ca. 70% epimer [100 mg, 0.26 mmole, 56% isolated yield basedon 6-hydroxyl-3-MEM-naloxol (2)].

¹H NMR (δ, ppm, CDCl₃): 6.50-6.73 (2H, multiplet, aromatic proton ofnaloxol), 5.78 (1H, multiplet, olefinic proton of naloxone), 5.17 (2H,multiplet, olefinic protons of naloxol), 4.73 (1H, doublet, C₅ proton ofα naloxol), 4.57 (1H, doublet, C₅ proton of β naloxol), 3.91 (1H,multiplet, C₆ proton of α naloxol), 3.51-3.75 (4H, multiplet, PEG), 3.39(3H, singlet, methoxy protons of PEG, α epimer), 3.36 (3H, singlet,methoxy protons of PEG, β epimer), 3.23 (1H, multiplet, C₆ proton of βnaloxol), 1.46-3.22 (14H, multiplet, protons of naloxol).

Example 6 Synthesis of —OCH₂CH₂(α,β-PEG₃-Nal)

6.a. Synthesis of an α and β Epimer Mixture of6-CH₃—(OCH₂CH₂)₃—O-3-MEM-Naloxol

NaH (60% in mineral oil, 38 mg, 0.94 mmole) was added to a solution of3-MEM-naloxol [98 mg, 0.24 mmole, from Example 5 and shown as (2) in theschematic therein] in dimethylformamide (“DMF,” 8 mL). The solution wasstirred at room temperature under an atmosphere of N₂ for 15 minutes, towhich was added a solution of CH₃—(OCH₂CH₂)₃Br (320 mg, 1.41 mmole) inDMF (1 mL). The resulting solution was then heated under N₂ in an oilbath for 2 hours.

HPLC analysis revealed that the desired product, a mixture of α- andβ-6-CH₃—(OCH₂CH₂)₃—O-3-MEM-naloxol was formed in approximately 95%yield. DMF was removed by a rotary evaporation to yield a sticky whitesolid. The crude product was used without further purification.

6.B. Synthesis of α and β Epimer Mixture of 6-CH₃—(OCH₂CH₂)₃—O-Naloxol(α,β-PEG₃-Nal)

The crude α- and β-6-CH₃—(OCH₂CH₂)₃—O-3-MEM-naloxol mixture from 6.A.above was dissolved in 3 mL of CH₂Cl₂ to form a cloudy solution, towhich was added 4 mL of trifluoroacetic acid (“TFA”). The resultingsolution was stirred at room temperature for 4 hours. HPLC analysisshowed that the reaction was complete. The solvent, CH₂Cl₂, was removedby a rotary evaporation. To the remaining solution was added 5 mL ofwater, followed by addition of K₂CO₃ to destroy excess TFA and adjustthe pH to 8. The solution was then extracted with CHCl₃ (3×50 mL). TheCHCl₃ extracts were combined and extracted with 0.1 N HCl solution (3×50mL). The remaining water phase was again adjusted to a pH of 8 byaddition of K₂CO₃, followed by extraction with CHCl₃ (3×50 mL). Thecombined organic extracts were then dried over Na₂SO₄. Following removalof the solvents, a colorless sticky solid was obtained.

The solid was purified by passage through a silica gel column (2 cm×30cm) twice using CHCl₃/CH₃OH (30:1) as the eluent. The purified product,a mixture of the α and β epimers of 6-CH₃—(OCH₂CH₂)₃—O-naloxolcontaining about equal amounts of the α and β epimers, was characterizedby NMR. (46 mg, 0.097 mmole, 41% isolated yield based on6-hydroxyl-3-MEM-O-naloxone). ¹H NMR (δ, ppm, CDCl₃): 6.49-6.72 (2H,multiplet, aromatic proton of naloxol), 5.79 (1H, multiplet, olefinicproton of naloxol), 5.17 (2H, multiplet, olefinic protons of naloxol),4.71 (1H, doublet, C₅ proton of α naloxol), 4.52 (1H, doublet, C₅ protonof naloxol), 3.89 (1H, multiplet, C₆ proton of α naloxol), 3.56-3.80(12H, multiplet, PEG), 3.39 (3H, singlet, methoxy protons of PEG, αepimer), 3.38 (3H, singlet, methoxy protons of PEG, β epimer), 3.22 (1H,multiplet, C₆ proton of β naloxol), 1.14-3.12 (14H, multiplet, protonsof naloxol).

6.C. Separation of α-6-CH₃—(OCH₂CH₂)₃—O-naloxol andβ-6-CH₃—(OCH₂CH₂)₃—O-naloxol

About 80 mg of a crude mixture of α and β epimers of PEG₃-Nal wasdissolved in a minimum of CHCl₃ and loaded onto a silica gel column (2cm×30 cm) prepared using CHCl₃. The column was carefully eluted with aCHCl₃/CH₃OH mixture (60:1). Pure α-PEG₃-Nal was the first-elutingspecies (26 mg, 33% isolated yield), followed by pure β-PEG₃-Nal (30 mg,38% isolated yield). Both compounds were colorless sticky solids.α-PEG₃-Nal, ¹H NMR (δ, ppm, CDCl₃): 6.49-6.73 (2H, two doublet, aromaticproton of naloxol), 5.79 (1H, multiplet, olefinic proton of naloxol),5.17 (2H, triplet, olefinic protons of naloxol), 4.71 (1H, doublet, C₅proton of naloxol), 3.81 (1H, multiplet, C₆ proton of naloxol),3.57-3.80 (12H, multiplet, PEG), 3.40 (3H, singlet, methoxy protons ofPEG), 1.13-3.12 (14H, multiplet, protons of naloxone). β-PEG_(S)-Nal, ¹HNMR (δ, ppm, CDCl₃): 6.54-6.72 (2H, two doublet, aromatic proton ofnaloxol), 5.77 (1H, multiplet, olefinic proton of naloxol), 5.15 (2H,triplet, olefinic protons of naloxol), 4.51 (1H, doublet, C₅ proton ofnaloxol), 3.58-3.78 (12H, multiplet, PEG), 3.39 (3H, singlet, methoxyprotons of PEG), 3.20 (1H, multiplet, C₆ proton of naloxol), 1.30-3.12(13H, multiplet, protons of naloxol).

α,β-6-CH₃—(OCH₂CH₂)₅—O-naloxol (“α,β-PEG₅-Nal”) andα,β-6-CH₃—(OCH₂CH₂)₇—O-naloxol (“α,β-PEG₇-Nal”) were similarly prepared,and their individual isomers separated and isolated.

Example 7 Oral Bioavailability of PEG-Mers of Cis-Retinoic Acid andNaloxol

Female Sprague Dawley® rats (150-200 g) were obtained from Harlan Labs.They were cannulated in the external jugular vein and allowed at least72 hours of acclimatization before the start of the study. The animalswere fasted overnight (day −1), but water was provided ad libitum.

On the morning of dosing (day 0), each rat was weighed and the cannulasflushed with heparin (1000 U/mL). With the aid of a feeding tube, theanimals were then dosed orally (gavage) with aqueous formulationscontaining either the PEGylated or the free drug. The dose wasdetermined on a mg/kg body weight basis. The total volume of the dosedid not exceed 10 mL/kg. At specific time intervals (1, 2 and 4 hours),blood samples (approximately 1.0 mL) were removed through the cannula,placed in 1.5 mL centrifuge tubes containing 14 μL of heparin, mixed andcentrifuged to separate the plasma. The plasma samples were frozen(<−70° C.) until assayed. The plasma samples were purified by aprecipitation technique and the analyte extracted and assayed using ahigh performance liquid chromatography (LC) method with a mass selectivedetector (MSD). Standard samples were prepared in the same way to createa standard curve, from which the concentration of unknown samples couldbe extrapolated (see results in Table II). When appropriate, an internalstandard was used in the analysis.

Selected properties of the tested compounds (such as the molecularweight and solubility) are summarized in Table I. The in-vitro enzymebinding activity of some of the tested compounds are also reported asIC₅₀ values in Table 1

TABLE 1 Selected Properties of Tested Compounds Molecular IC₅₀ DrugWeight Solubility (μM) (nM)* 13-cis-Retinoic Acid 300.45 0.47 — (parentdrug) PEG₃-13-cis-RA 445.64 3.13 — PEG₅-13-cis-RA 549.45 soluble —PEG₇-13-cis-RA 621.45 58.3  — PEG₁₁-13-cis-RA 797.45 soluble — Naloxone“Nal” 327.37 soluble as HCl salt 6.8 (parent drug) α isomer of PEG₃-Nal475.6 soluble 7.3 β isomer of PEG₃-Nal 475.6 soluble 31.7 α isomer ofPEG₅-Nal 563.0 soluble 31.5 β isomer of PEG₅-Nal 563.0 soluble 43.3 αisomer of PEG₇-Nal 652.0 soluble 40.6 β isomer of PEG₇-Nal 652.0 soluble93.9 α isomer of PEG₉-Nal 740.0 soluble 64.4 β isomer of PEG₉-Nal 740.0soluble 205.0 Hydroxyzine “Hyd” 374.91 soluble as HCl salt 48.8 (parentdrug) PEG₁-Hyd 433.0 soluble 70.3 PEG₃-Hyd 521.0 soluble 105.0 PEG₅-Hyd609.0 soluble 76.7 Cetirizine “Cet” 388.89 soluble as HCl salt 77.1(parent drug) PEG₁-Cet 446.0 soluble 61.0 PEG₃-Cet 534.0 soluble 86.4PEG₅-Cet 622.0 soluble 128.0 *Mu-opiate binding activity for naloxoneseries of compounds Histamine H-1 binding activity for hydroxyzine andcetirizine series of compounds

The oral bioavailabilities of the retinoic acid series of compounds werecalculated and the results provided in Table II. All the data wasnormalized to a 6 mg/kg dose. The plasma concentration versus timeprofiles for these compounds are provided in FIG. 1.

TABLE II Oral Bioavailabilities of the Retinoic Acid Series of CompoundsMean Plasma Concentration (ng/mL) ± SD N Drug 1 hr 2 hr (rats)13-cis-Retinoic Acid 43.3 ± 24.0 23.3 ± 14.8 3 PEG₃-13-cis-RA 131.8 ±55.0  158.0 ± 133.0 7 PEG₅-13-cis-RA 77.7 ± 31.6 61.6 ± 57.1 4PEG₇-13-cis-RA 44.0 ± 13.0 38.7 ± 4.2  3 PEG₁₁-13-cis-RA 21.8 ± 7.1 58.2 ± 43.5 4

The oral bioavailability of each isomer in the naloxone series ofcompounds was calculated and is provided in Table III. The oral naloxonedose was either 5 or 10 mg/kg and the doses for the PEGylated compoundswere normalized to 1 mg/kg dose. The plasma concentration versus timeprofiles for these compounds is provided in FIG. 2.

TABLE III Oral Bioavailabilities of the Naloxone Series of CompoundsMean Plasma Concentration (ng/mL) ± SD N Drug 1 hr 2 hr (rats) Naloxone3.67 ± 1.05  3.11 ± 0.46 4 α-PEG₃-Nal 37.28 ± 4.99  14.92 ± 5.27 5β-PEG₃-Nal 53.79 ± 5.19  22.47 ± 8.78 5 α-PEG₅-Nal 27.37 ± 10.82 15.38 ±6.65 6 β-PEG₅-Nal 69.34 ± 15.03  36.92 ± 15.84 5 α-PEG₇-Nal 40.08 ±16.61 39.51 ± 9.57 4 β-PEG₇-Nal 50.41 ± 36.44  50.08 ± 25.28 4

The above results show that PEGylation of small, lipophilic compoundslike retinoic acid and naloxone (the free base form) increases theirsolubility and oral bioavailability. On the other hand, attachment ofoligomeric PEGs also increases the molecular weight of the parentcompound (greater than about 500 Daltons), which in turn can restrictthe oral permeation of highly water soluble compounds, particularly withincreasing PEG-mer length, as seen for example with PEG₇-13-cis-RA andPEG₁₁-13-cis-RA.

Example 8 Transport Across the Blood Brain Barrier (BBB) of PEG-Mers ofCis-Retinoic Acid and Naloxone

As utilized for these experiments, the in situ brain perfusion techniqueemployed the intact rat brain to (i) determine drug permeation acrossthe BBB under normal physiological conditions, and (ii) to studytransport mechanisms such as passive diffusion verses carrier mediatedtransport.

Perfusion was performed using the single time-point method. Briefly, theperfusion fluid (perfusate) containing the test compound(s) was infusedinto rats via the left external carotid artery at a constant rate by aninfusion pump (20 mL/min). Perfusion flow rate was set to completelytake over fluid flow to the brain at normal physiologic pressure (80-120mm Hg). The duration of the perfusion was 30 seconds. Immediatelyfollowing the perfusion, the brain vasculature was perfused for anadditional 30 seconds with drug-free perfusate to remove residual drug.The pump was turned off and the brain was then immediately removed fromthe skull. Left-brain samples from each rat were first weighed and thenhomogenized using a Polytron homogenizer. Four (4) mL of 20% methanolwas added to each rat brain for homogenization. After homogenization,the total volume of homogenate was measured and recorded.

A measured amount of the homogenate was diluted with organic solvent andsubsequently centrifuged. The supernatant was removed, evaporated in astream of nitrogen and reconstituted and analyzed by LC/MS/MS.Quantification of drug concentrations in brain homogenate was performedagainst calibration curves generated by spiking the drugs into blank(i.e. drug-free) brain homogenate. Analysis of the drug concentrationsin brain homogenates was carried out in triplicate, and the values wereused to calculate the brain uptake rate in pmole per gram of rat brainper second of perfusion.

Each perfusion solution contained atenolol (target concentration, 50μM), antipyrine (target concentration, 5 μM) and a test compound(13-cis-retinoic acid, PEG_(n)-13-cis-retinoic acid, naloxone orPEG_(n)-Nal) at a target concentration of 20 μM.

The BBB uptake of each compound tested was calculated, normalized andrecorded in Table IV. All the data was normalized to a 5 μM dosingsolution at 20 mL/min perfusion rate for 30 sec.

TABLE IV Blood-Brain Barrier (BBB) Uptake for Tested CompoundsNormalized Brain Uptake Rate in pmole/gm brain/sec N Drug (Mean ± SD)(rats) Atenolol (low standard) 0.7 ± 0.9 4 Antipyrine (high standard)17.4 ± 5.7  4 13-cis-Retinoic Acid 102.54 ± 37.31  4 PEG₃-13-cis-RA79.65 ± 20.91 4 PEG₅-13-cis-RA 58.49 ± 13.44 3 PEG₇-13-cis-RA 24.15 ±1.49  3 PEG₁₁-13-cis-RA 17.77 ± 1.68  3 Naloxone 15.64 ± 3.54  3PEG₃-Nal 4.67 ± 3.57 3 PEG₅-Nal 0.96 ± 0.36 3 PEG₇-Nal (α isomer) 0.94 ±0.32 3 PEG₇-Nal (β isomer) 0.70 ± 0.19 3 Hydroxyzine 355.89 ± 59.02  3PEG₅-Hyd 131.60 ± 15.84  3 PEG₇-Hyd 12.01 ± 2.97  3 Cetrizine 1.37 ±0.37 3 PEG₅-Cet 4.32 ± 0.26 3 PEG₇-Cet 1.13 ± 0.05 3

The above results demonstrate that PEGylation of a lipophilic compoundsuch as 13-cis-retinoic acid can significantly reduce its brain uptakerate (“BUR”), e.g., by a factor of four in the case of PEG₇-13-cis-RA,and by a factor of five in the case of PEG₁₁-13-cis-RA as compared tothe parent compound “13-cis-retinoic acid”. In the case of naloxone, areduction in BUR of 16 times was observed for PEG₅-Nal and PEG₇-Nal.With respect to hydroxyzine, the BUR was reduced about 29 times whenadministered as PEG₇-Hyd. The relatively minimal transport of cetirizineacross the blood-brain barrier was not altered significantly whenadministered as PEG₇-Cet.

Thus, overall, it was surprisingly discovered that by attaching smallwater-soluble polymers to small molecule drugs such as these, one canoptimize a drug's delivery profile by modifying its ability to crossbiological membranes, such as the membranes associated with thegastro-intestinal barrier, the blood-brain barrier, the placentalbarrier, and the like. More importantly, it was discovered that, in thecase of orally administered drugs, attachment of one or more smallwater-soluble polymers is effective to significantly reduce the rate oftransport of such drugs across a biological barrier such as theblood-brain barrier. Ideally, the transport of such modified drugsthrough the gastro-intestinal tract is not adversely impacted to asignificant degree, such that while transport across the biologicalbarrier such as the blood-brain barrier is significantly impeded, theoral bioavailability of the modified drug is retained at a clinicallyeffective level.

The data generated in Examples 7 and 8 was plotted in order to comparethe effect of PEG size on the relative oral bioavailability and BBBtransport of 13-cis-retinoic acid and naloxone, respectively. See FIGS.3-7. In FIG. 3, the effect of attaching each of a PEG 3-mer, a PEG5-mer, a PEG 7-mer and a PEG 11-mer to 13-cis-retinoic acid on its oralbioavailability is examined. In FIG. 4, the effect of covalentattachment of these various PEG-mers on the blood-brain barriertransport of 13-cis-retinoic acid is examined. In FIG. 5, the effect ofcovalent attachment of each a PEG 3-mer, PEG 5-mer and a PEG 7-mer onthe oral bioavailability of naloxone is examined. FIG. 6 demonstratesthe effect of covalent attachment of such PEG-mers on the bloodbrain-barrier transport of naloxone. FIG. 7 shows that the PEG_(n)-Nalcompounds had a higher oral bioavailability than naloxone. As can beseen from these figures, as the size of the PEG oligomer increases, theBBB uptake rate significantly decreases, while the oral bioavailabilityincreases relative to that of the parent molecule.

The difference in oral bioavailability between the α- and β-isomers ofnaloxone may be due to the differences in their physicochemicalproperties. One isomer appears to be slightly more lipophilic than theother isomer, and thereby results in a small difference in oralbioavailability.

Example 9 In-Vitro Metabolism of PEG-Naloxol

An in vitro method was developed to study the effect of PEGylation onthe Phase II metabolism (glucuronidation) of naloxone. The procedurecalls for the preparation of a NADPH regenerating system (NRS) solution.The NRS solution is prepared by dissolving sodium bicarboante (22.5 mg)in 1 mL of deionized water. Into this solution B-nicotinamide adeninedinucleotide phosphate sodium salt or NADP (1.6 mg), glucose-6-phosphate(7.85 mg), glucose-6-phosphate dehydrogenase (3 μA), uridine5-diphosphoglucuronic acid trisodium salt or UDPGA (2.17 mg), adenosine3′-phosphate 5′-phosphosulfate lithium salt or PAPS (0.52 mg), and 1 Mmagnesium chloride solution (10 μL) were added. After the solids wereall dissolved, the solution was stored in an ice bath.

30 mM test article stock solutions were prepared by dissolving weighedamounts of naloxone HCl, 6-mPEG₃-O-Naloxone, α-6-mPEG₅-O-naloxone, andα-mPEG₇-O-Naloxone in 1 mL of deionized water.

Male Sprague Dawley rat microsomes (0.5 mL at 20 mg/mL concentration;M00001 from In-vitro Technologies, Baltimore, Md.) were removed from thefreezer and thawed in an ice bath. Forty μL of the liver microsomes werediluted to 100 μL with 60 μL of deionized water in a test vial. To thetest vial, tris buffer, pH 7.4 (640 μL) and a test article stock (10 μL)were added to have 750 μL volume.

Each test vial and the NRS solution were separately placed in a 37° C.water bath for 5 minutes. The NRS solution (250 μL) was added into eachtest vial. The reaction timer was started at the addition of the NRS tothe first test vial. Each sample (200 μL) was collected and thenperchloric acid (20 μL) was added to terminate the reaction. The sampleswere collected at the following time points: 0-2, 20, 40 and 60 minutes.All of the terminated test vials were stored in an ice bath.

Acetonitrile (100 μL) was added into each test vial, which was thencentrifuged at 3000×g for 5 minutes. Supernatant (230 μL) was withdrawnand then 10 μL of the test solution was assayed by an LC/MS method. Theconcentration of test article in each sample was measured and recordedat each time point.

Table V lists the percentage of active remaining after incubation withliver microsomes.

TABLE V Percentage of Active Remaining After Incubation with LiverMicrosomes Time α-PEG₃- β-PEG₃- α-PEG₅- α-PEG₇- (min) naloxone Nal NalNal Nal 0 100.0 100.0 100.0 100.0 100.0 20 47.1 64.8 83.9 84.1 87.4 4027.6 51.7 75.2 75.6 81.6 60 15.6 45.7 69.6 69.2 76.9

In view of the results in Table V, it is possible to conclude thatPEGylation with an oligomer decreases that rate of glucuronidation for asmall molecule such as naloxol. Furthermore, as the PEG oligomer chainincreases, the rate of glucuronidation decreases. In addition,comparison of α-isomers and β-isomers of PEG₃-naloxol, shows that theβ-isomer is a poor substrate for cytochrome P450 isozymes in theisolated rat liver microsomes. This observation confirms the in-vivodata illustrated in FIG. 7.

Turning to the data in FIGS. 8 and 9, it appears that attachment ofsmall PEGs can be effective in decreasing the rate of drug metabolism(as indicated by glucuronide formation in the case of naloxone). Thehigher levels of the 13-isomer in the blood when compared to theα-isomer is likely due to a significant prevention of the first passeffect, that is to say, a significant prevention of the extent of firstpass metabolism (FIG. 7), resulting from covalent attachment of theoligomeric PEG molecule. The PEG molecule may create steric hinderanceand/or hydrophilic or hydrophobic effects, which when the PEG isattached to the β-isomer form, alters the affinity of the β-isomerconjugate to cytochrome P450 isozymes to a greater degree than when thePEG is attached to the α-isomer form. The levels of β-isomer metaboliteare lower when compared to the α-isomer metabolite and unPEGylatednaloxone.

Example 10 Activity of Various Opiod Antagonists on μ-Opiate Receptors

In a separate series of experiments, the bioactivity of naloxone, otheropiod antagonists, and various conjugates on μ-opiate receptors wasdetermined in-vitro. The results are summarized in Table VI.

TABLE VI Activity of Naloxone and PEG_(n)-6-Naloxol Conjugates onμ-Opiate Receptors, in-vitro. Molecular EC₅₀ Compound Weight (nM)Naloxone 327.4 6.8 3-PEG₃-O-naloxone 474 2910.0 6-NH₂-naloxone 601 29.2PEG₅₅₀-6-NH-naloxone (PEG₁₃ amide) 951 210.0 α-6-naloxol 329 2.0β-6-naloxol 329 10.8 α-PEG₃-Nal 475.6 7.3 β-PEG₃-Nal 475.6 31.7α-PEG₅-Nal 563 31.5 β-PEG₅-Nal 563 43.3 α-PEG₇-Nal 652 40.6 β-PEG₇-Nal652 93.9

In the table above, for each compound, the bioactivity is provided as ameasure of the relative bioactivity of each of the various PEGconjugates in comparison to parent drug. The EC₅₀ is the concentrationof agonist that provokes a response halfway between the baseline andmaximum response in a standard dose-response curve. As can be seen fromthe above data, each of the PEG_(n)-Nal conjugates is bioactive, and infact, all of the 6-naloxone or naloxol conjugates maintained a degree ofbioactivity that is at least 5% or greater than that of the parent drug,with bioactivities ranging from about 5% to about 35% of the bioactivityof the unmodified parent compound. In terms of bioactivity,PEG₅₅₀-6-NH-naloxone possesses about 13% of the bioactivity of theparent compound (6-NH₂-naloxone), α-PEG₃-Nal possesses about 30% of thebioactivity of the parent compound (α-6-OH-naloxol), and β-PEG₃-Nalpossesses about 35% of the bioactivity of the parent compound(α-6-OH-naloxol).

Example 11 Method of Making Substantially Unimolecular WeightOligo(Ethylene Glycol) Methyl Ethers and their Derivatives

The unimolecular (monodisperse) PEGs of the present invention wereprepared as set forth in detail below. These unimolecular PEGs wereparticularly advantageous in providing the modified active agents of thepresent invention, and in imparting the desired modification of barriertransport properties of the subject active agents.

The method exemplified below represents another aspect of the presentinvention, that is, a method for preparing monodisperse oligo(ethyleneoxide) methyl ethers from low molecular weight monodisperseoligo(ethylene glycol)s using halo-derivatized (e.g., bromo derivatized)oligo(ethylene oxide). Also provided herein, in another aspect of theinvention, is a method of coupling oligo(ethylene oxide) methyl ether(from a unimolecular weight composition) to an active agent using ahalo-derivatized oligo(ethylene oxide) methyl ether.

Schematically, the reaction can be represented as follows:

10.A. Synthesis of CH₃O—(CH₂CH₂O)₅—H with CH₃OCH₂CH₂Br

Tetra(ethylene glycol) (55 mmol, 10.7 g) was dissolved in 100 mL oftetrahydrofuran (“THF”) and to this solution was added KOtBu (55 mL,1.0M in THF) at room temperature. The resulting solution was stirred atroom temperature for 30 minutes, followed by dropwise addition ofCH₃OCH₂CH₂Br (55 mmol, 5.17 mL in 50 mL THF). The reaction was stirredat room temperature overnight, followed by extraction with H₂O (300mL)/CH₂Cl₂ (3×300 mL). The organic extracts were combined and then driedover anhydrous Na₂SO₄. After filtering off the solid drying agent andremoving the solvent by evaporation, the recovered crude residue waspurified by column chromatography using a silica gel column (CH₂Cl₂:CH₃OH=60:1˜40:1) to give pure penta(ethylene glycol) monomethyl ether(yield 35%). ¹H NMR (CDCl₃) δ 3.75-3.42 (m, 20H, OCH₂CH₂O), 3.39 (s, 3H,MeO).10.B. Synthesis of CH₃O—(CH₂CH₂O)₇—H using MeOCH₂CH₂Br

To a solution of hexa(ethylene glycol) (10 g, 35 mmole) and 2-bromoethylmethyl ether (4.9 g, 35 mmole) in THF (100 mL) was slowly added sodiumhydride (2.55 g, 106 mmole). The solution was stirred at roomtemperature for two hours. HPLC indicated that mPEG₇-OH was formed inabout 54% yield. The reaction was then stopped by the addition ofdiluted hydrochloride acid to destroy excess sodium hydride. Allsolvents were removed using a rotary evaporator to give a brown stickyliquid. Pure mPEG₇-OH was obtained as a colorless liquid (4.9 g, 41%isolated yield) by using semi-preparative HPLC (20 cm×4 cm, C18 column,acetonitrile and water as mobile phases). ¹H NMR (CDCl₃): 2.57 ppm(triplet, 1H, OH); 3.38 ppm (singlet, 3H, CH₃O); 3.62 ppm (multiplet,30H, OCH₂CH₂).

10.C. Synthesis of CH₂CH₂O)₅—Br

Triethyl amine (5.7 ml, 40 mmol) was added to CH₃O—(CH₂CH₂O)₅—OH (5.0 g,20 mmol) with stirring. The solution was cooled in an ice bath under N₂,and 2.5 ml of methanesulfonyl chloride (32 mmol) was added dropwise over30 minutes. The solution was then stirred overnight at room temperature.Water (40 ml) was added to the reaction mixture and the solution wasextracted with CH₂Cl₂ (3×150 ml) and the organic phase was washed with0.1 N HCl (3×80 ml) and water (2×80 ml). After drying with Na₂SO₄ andremoval of solvent, a light brown liquid was obtained. The product andBu₄NBr (12.80 g, 39.7 mmol) were dissolved in CH₃CN (50 ml), and theresulting solution was stirred under N₂ at 50° C. for 15 hours. Aftercooling to room temperature, CH₃CN was removed by rotary evaporation togive a red liquid, which was dissolved in 150 ml water and extractedwith EtOAc (2×200 ml). The organic phase was combined, washed withwater, and dried over Na₂SO₄. After the removal of solvent, a red liquidwas obtained (4.83 g, 77.4%). ¹H NMR (300 Hz, CDCl₃): δ 3.82 (t, 2H),3.67 (m, 14H), 3.51 (m, 2H), 3.40 (s, 3H).

Synthesis of mPEG3 N-Mefloquine

To a methanol solution (5 mL) of mefloquine HCl salt (200 mg, 0.48 mmol)and mPEG₃-Butyaldehyde (280 mg, 1.20 mmol) was added sodiumcyanoborohydride (60 mg, 0.96 mmol) water solution (1 mL). The resultingsolution was heated under nitrogen with stirring in an oil bath at 50°C. for 16 hours. HPLC showed that the reaction was complete. Allsolvents were then removed by a rotary evaporator to give a crudeproduct. After purified by a preparative reverse phase HPLC, puremPEG-3-N-Mefloquine conjugate was obtained as a colorless sticky liquid(160 mg, 0.27 mmol, 56% isolated yield). ¹H NMR (CDCl₃, ppm): 8.15(multiplet, 3H, aromatic ring); 7.73 (triplet, 1H, aromatic ring); 5.86(doublet, 1H, CH); 3.67 (multiplet, 14H, PEG back bone); 3.52 (singlet,3H, PEG-OCH₃); 3.18 (multiplet, 2H, PEG-CH₂); 0.52-2.74 (multiplet, 13H,PEG and cyclohexyl protons).

Schematically, the reaction can depicted as follows:

Example 12 Preparation of PEG-FITC Molecules and Experimental Protocols12.A—PEG-FITC Conjugates

A series of PEG-fluorescein isothiocynate (FITC) molecules were preparedwhereby the different sized PEGs were covalently attached via thethiocyanate functionality on the FITC resulting in a FITC-thiourea-PEGconjugate. Use of the mPEGn-OH reagent in the conjugation reaction forPEG sizes 0.55K, 1K, 2K, 5K and 20K Daltons ensured a 1:1 ratio of PEGto FITC, i.e. each PEG-FITC conjugate consisted of one FITC molecule perPEG molecule. However, the conjugate composed of 3.4 K-PEG-FITC wasgenerated using the HO-PEGn-OH diol reagent, which allows for thepossibility of two different species, either two FITC (a “dumbbell”) orone FITC molecule per PEG. Since unconjugated FITC is highly reactiveand would bind to many proteins in tissues and cells, sodium fluoresceinwas employed as a control, to determine the residence time of the parentnon-PEGylated molecule in the lung.

PEG was conjugated with FITC. Note that in the case of PEG(OH) FITC (the3.4K PEG-FITC species), the methyl group at the end of the PEG chainwould be replaced by a hydroxyl group, or by a second FITC moiety. Thestructure of the PEG-FITC conjugate is shown below.

12.B—Design of Animal Experiments

To determine the effect of PEG length on residence time in the lung, aseries of in vivo experiments was performed in which each PEG-FITCspecies was administered intratracheally (IT) to rats, and the amountremaining in the lung was determined at progressive time points. Foreach species, 30 μg PEG-FITC, prepared in PBS/0.5% BSA, was administeredIT to rats (300 μL of a 100 μg/mL solution). Animals were sacrificed intriplicate at the following time points: predose, 10 min (0.17 h), 1 h,3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 168 h (7 days), and subjected tobronchoalveolar lavage (BAL) with a single wash, using 0.9% NaCl (154 mMNaCl—physiological saline concentration). Following the lavage, lungswere excised, and samples of serum were extracted. The resulting lavagesolution was then centrifuged to pellet the cellular fraction (BALcells) and separate it from the BAL supernatant. An outline of theapproach is shown in FIG. 11.

12.C—Quantitation of Test Molecules in Biological Matrices

Quantitation of PEG-FITC concentrations in the various compartments wasperformed using a Tecan™ Genios Pro microplate reader to detect thefluorescence intensity of the samples under conditions suitable fordetection of fluorescein (λex 485 nm, λem 535 nm). Samples from BAL, BALcells and serum were read directly in microtiter plates with no priortreatment. To quantitate PEG-FITC remaining in the residual lungmaterial, however, the excised lungs were homogenized and PEG-FITC wasextracted in ethanol.

For all samples, quantitation was performed by interpolation from astandard curve generated from known concentrations of the relevantmolecule prepared in the correlating matrix. Thus for samples of BAL andBAL cells, the standard samples were prepared in 0.9% NaCl, for serumsamples, the standards were prepared in commercially obtained rat serum,and for lung extracts, the standard samples were prepared in PBS:Ethanol(1:3). Lower limits of quantitation for each molecule tested in therespective matrices is shown in Table VII below.

TABLE VII Lower limits of quantitation for PEG-FITC molecules. PBS:EtOHMolecule BAL (0.9% NaCl) Serum (lung extract) Sodium fluorescein 2.5nM   0.9 ng/mL  4 nM 1.5 ng/mL  0.24 nM 0.09 ng/mL 0.55K-PEG-FITC 5 nM 4 ng/mL ND ND  0.5 nM  0.4 ng/mL 1K PEG-FITC 5 nM  6 ng/mL ND ND 0.24nM 0.27 ng/mL 2K PEG-FITC 5 nM 12 ng/mL 8 nM 18 ng/mL 0.24 nM 0.55 ng/mL3.4K PEG-FITC 5 nM 18 ng/mL 8 nM 30 ng/mL   2 nM   7 ng/mL 5K PEG-FITC 5nM 30 ng/mL 4 nM 25 ng/mL 0.24 nM  1.5 ng/mL 20K PEG-FITC 5 nM 110ng/mL  ND ND 0.24 nM   5 ng/mL ND—not determined.

12.D—Data Analysis

Concentrations of PEG-FITC were interpolated from a standard curve, andwere analyzed using GraphPad Prism 4.01 software and Microsoft Excel.For BAL studies, the total amount remaining was calculated for eachanimal from the product of the concentration and the BAL volumerecorded. For BAL cells, the pellet was resuspended in 1 mL; the amountpresent was calculated accordingly. For residual lung tissue, the amountpresent in both lungs (μg/lung) was calculated from the product of theconcentration and the homogenate volume. Note that although the nominaldose delivered in each experiment was 30 μg, the dosing solution wasanalyzed from the same standard curve for each experiment and the actualdelivered dose was calculated. The data shown throughout thisspecification are thus corrected for each PEG-FITC species respectivelyand are represented as percentage dose administered. The doses deliveredin each experiment are represented in Table VIII below.

TABLE VIII Pharmacokinetic data from in vivo experiments. BAL BAL BALBAL Lung Dose Molecule MW k-el t_(1/2)-el t₉₀ t₉₉ t_(1/2)-el (μg) Sodium376 1.3253 0.523 1.74 3.5 0.5021 40 fluorescein 0.55K-PEG- 810 0.29322.364 7.85 15.7 2.351 50 FITC 1K PEG- 1143 0.1391 4.984 16.56 33.1 5.17945 FITC 2K PEG- 2303 0.0968 7.161 23.79 47.6 7.595 50 FITC 3.4K PEG-3655 0.0770 9.005 29.91 59.8 13.08 40 FITC 5K PEG- 6149 0.0577 12.0239.93 79.9 11.8 53 FITC 20K PEG- 21930 0.0551 12.57 41.76 83.5 12.51 55FITC

Example 13 BAL Supernatant

The concentration of PEG-FITC remaining in BAL supernatant was plottedversus time for each PEG-FITC species as shown in FIG. 12. Theconcentrations were determined in accordance with the protocols inExample 12, and the concentration-time relationship of each PEG-FITCspecies in BAL is expressed as percentage dose. Analysis of the amountof PEG-FITC remaining in the BAL supernatant at increasing time pointsrevealed that each PEG-FITC species was eliminated from the BAL in amanner described by a single exponential curve on a concentration-timecurve. As the molecule's PEG chain length increases, its lung residencetime becomes more prolonged, as indicated by the increased half-life forelimination from BAL (t_(1/2)-el), as shown in FIG. 13. The eliminationhalf life (t_(1/2)-el) determined from the concentration-time plots inFIG. 12 is plotted against molecular weight (MW), and produces anexponential association relationship, described by the equation:t_(1/2)-el=12.84*(1-e^(−kMW)), where k=0.000357, MW=molecular weight inDaltons, and t_(1/2)=elimination half-life in hours.

Conjugation to FITC of a PEG as small as even 0.55 K causes an extensionin residence time, the t1/2-el increasing from <0.52 h to 2.36 h. Thet1/2-el calculated for sodium fluorescein (0.52 h) is an overestimate,since its elimination from the lung was so rapid that at the first timepoint (10 min) less than 50% of the delivered drug was recovered. Forall the PEG conjugated species, by contrast, the majority of thedelivered dose was recovered in the BAL at the earliest time point. Withthe exception of 3.4K-PEG-FITC, over 80% of the dose administered wasrecovered in BAL 10 min following IT administration. In the case of3.4K-PEG(OH)-FITC, approximately 60% of the initial dose is recovered at10 min, and this value remains relatively unchanged at the next timepoint, 3 h. The reason for this is not clear, but the calculatedconcentration arises from readings taken from three animals, suggestingthat technical error is unlikely. It is possible that the PEG(OH)reagent is more reactive with endogenous molecules than the mPEG whichis conjugated to FITC in the other species, and that this alters theinitial absorption through the lung. Alternatively, the presence of twoFITC moieties per molecule, as per the “dumbbell” configuration mayalter the absorption or the detection characteristics of this PEG-FITCspecies. In all cases, the PEG-FITC was completely eliminated from BAL,although the time at which this occurred increased with PEG size; thatis, at later time points, PEG-FITC concentrations in the single BAL washfell below the lower limit of quantitation, and represented aninsignificant portion of the delivered dose.

A clear relationship exists between t_(1/2)-el from BAL and molecularweight (FIG. 13). The plot shows that the maximum t_(1/2)-el generatedby PEG is 12.8±0.6 h, and suggests that, for controlling the rate ofelimination from the lung, the most effective range of PEG size toemploy extends to approximately 3.4 K, with the maximum t_(1/2)-elreached at 5K; beyond this, therefore, there is little to gain inresidence time by increasing the size of PEG. Based on the eliminationrates calculated from the data above estimations can be made regardingthe maximum time that the molecules can be retained in the lung. The t₉₀and t₉₉ values represented in Table VIII demonstrate the time at which90% and 99% respectively of the dose of each species would beeliminated. For example, for 2K-PEG-FITC, 90% of the dose is eliminatedfrom BAL after approximately 24 h (23.8 h). It appears that the maximumextension of residence time that PEG can endow on FITC molecule is inthe order of 1-2 days. If so, the equation described in FIG. 13 can beused to predict what size PEG should be conjugated to a small moleculeto achieve a specific residence time in the lung, and can serve todirect decisions for candidate drug molecules.

The BAL compartment derives from a single wash of the lungs, andtherefore contains molecules and cells that reside on the “airside”(lumen) of the pulmonary epithelium, in addition to those that areloosely attached to cell membranes. PEGylated compounds detected in thisfraction therefore represent drug molecules that would be available foraction at an extracellular target in the lung, such as those that targetcell surface receptors (e.g., β-agonists used in the treatment ofasthma) or that target micro-organisms that infect the lung (e.g.,anti-infectives). Conversely, for systemic targets, where the lung isused as a depot, PEGylation could serve as a means to slow theabsorption through the lung.

Example 14 BAL Cells

The amount of PEG-FITC detected in the BAL cells fraction at each timepoint is displayed in FIG. 14. The percentage of dose that associateswith the BAL cells' fraction is plotted against time for each PEG-FITCspecies. The cellular fraction of BAL constitutes primarily alveolarmacrophages and contains a minor amount of epithelial cells that mayhave become detached in a single wash. The association of PEG-FITCmolecules with this fraction most likely, therefore, represents aclearance mechanism operating by alveolar macrophages to eliminateforeign material present in the lung. Most notable among the plots inFIG. 14 is the different behavior of the smaller PEG-FITC molecules(0.55K and 1K) from that exhibited by the larger ones (2K, 3.4K, 5K,20K). Whereas the smaller PEG-FITC molecules are fully eliminated andshow a single phase of elimination, the larger PEG-FITC moleculesdisplay a biphasic response. For these molecules an initial rise isseen, followed by a decrease at 24 h; subsequently a second phase isseen with an increase in the association of PEG-FITC in BAL cells.

One possible explanation for the observed profiles is that the longerresidence of the larger PEG-FITC molecules triggers a response inmacrophages that recruits additional phagocytes from the systemiccirculation to remove the foreign material. The smaller PEG-FITCspecies, by contrast, do not remain in the lung for long enough totrigger such a response. It is worth noting that no evidence ofphagocytosis is presented, nor any identification of the identities ofthe cell types with which the PEG-FITC associates. Alternatively, it ispossible that the uptake of PEG-FITC is not the result of an activephagocytosis triggered by the presence of PEG-FITC, but rather is due anon-specific pinocytotic mechanism, caused by the fluid influx from theintratracheal procedure. PEG-FITC remaining in the lung would, in thisscenario, be taken up along with the fluid. The increased residence ofthe larger PEG-FITC molecules would underlie the differentialassociation with macrophages exhibited by these species.

FIG. 15 demonstrates the relative uptake of the different PEG-FITCmolecules on the same ordinate scale. A few interesting features appearin this comparison. First, the non-PEGylated parent molecule, sodiumfluorescein, demonstrates a relatively high association with BAL cellsat the earliest time point (10 min) compared with all the PEGylatedspecies. This may indicate that a significant portion (approximately13%) of the unmodified molecule is recognized and cleared by macrophagesimmediately upon delivery to the lung. The addition of PEG, even ofsmall chain-length, appears to prevent this rapid uptake, and mightcontribute the ability of PEG to prolong the residence of thesemolecules in the epithelial lining fluid. Future studies will addresswhether this phenomenon is exhibited by different classes of drugmolecules when conjugated to PEG.

Second, at 48 h post dose, a significant portion of the delivered doseof the larger molecules (PEG sizes 2K, 3.4K, 5K and 20K) associates withthe presumed alveolar macrophages. Furthermore, for as long as 7 days(168 h) post-administration, 5-10% of the dose still remains associatedwith this fraction for the 5K and 20K PEG-FITC molecules. This suggeststhat the larger sized PEG molecules might be able to reside for extendedperiods in the alveolar macrophages, and may be able to protect theFITC, which is the moiety detected in the quantitative assay, fromdegradation. While evidence is not presented demonstrating the integrityof the FITC-PEG molecule in these cells, the differential profiles ofthe non-PEGylated and PEGylated species suggests that the conjugatedmolecule remains intact. Indeed, if the PEG had cleaved from the FITC, arapid clearance such as that seen with the non-PEGylated molecule wouldbe expected.

Third, a clear dependence of percentage uptake on PEG-size appears.Thus, the actual percentage of the delivered dose that remainsassociated with BAL cells at later time points increases with PEG chainlength. This could arise from the extended residence time of theselarger molecules. Thus, the longer the molecule remains in the lung, thegreater the probability that it will associate with alveolar macrophagesor other phagocytes which may have been recruited. The experimentaldesign employed here, however, cannot distinguish between macrophagesthat associate with PEG-FITC at an earlier time point and retain it for7 days from macrophages which may have taken up the molecule on day 7.

Example 15 Residual Lung Material

Following the BAL procedure outlined in Example 12, the remaining lungswere excised and PEG-FITC concentrations were determined from extractsof the homogenates. FIG. 16 illustrates the percentage of the dose thatremains in the residual lung material plotted against time for eachPEG-FITC species.

The residual lung material extracted following the BAL procedureconsists of: (i) epithelial lining fluid and alveolar macrophages thatwere not fully extracted during the single wash of BAL, (ii) a smallamount of blood that remains in the lung and (iii) the lung tissue.

The pattern of elimination of PEG-FITC from the residual lung materialresembles that seen with the BAL cells' fraction. Thus for the smallerPEG sizes (0.55K, 1K) complete elimination is seen (by 12 h and 18 hrespectively). However, for the larger PEG sizes (2K, 3.4K, 5K, 20K) adifferent profile appears, with approximately 10-15% of the doseremaining associated with the residual lung tissue at extended times.Note that for 2K and 3.4K PEG sizes, data is only available up to 48 h(2 d) post administration. For the 5K and 20K PEG sizes, data from 168 h(7 d) post dose shows a significant amount still associated with lungtissue. Since no PEG-FITC remains in the BAL at this time point, andserum concentrations lie below detectable levels, it seems likely thatthe majority of the material at the 168 h time point lies in the lungtissue itself, with perhaps some also associated with macrophages in thelungs.

Example 16 Serum Concentrations of PEG-FITC

Serum concentrations of PEG-FITC were analyzed as described in Example12. FIG. 17 shows the concentration-time relationship of PEG-FITC inserum for three PEG-FITC species. For all PEG-FITC species,concentrations in serum remained so low that they were at or close tothe lower limit of quantitation at all time points; these limits lie atunder 1% of the administered dose. Only sodium fluorescein, 10 min postdose, produced a peak in serum that could easily be measured. Based on arat serum volume of approximately 8 mL the peak for fluorescein wouldlie at approximately 2 μg, which represents less than 10% of theadministered dose. By contrast, concentrations at all other time points,and for all other molecules, represent negligible proportions of theadministered dose.

No analysis of whole blood was performed. Preliminary evidence suggeststhat the PEG-FITC molecules do not partition heavily into blood comparedwith serum (data not shown). Evidence in the literature indicates thatPEG molecules in the size range employed here are readily cleared fromserum by the kidneys. It seems likely, therefore, that PEG-FITC that isabsorbed from the lungs is cleared so rapidly by the kidneys that serumconcentrations remain very low. Such a scenario suggests a model offlip-flop kinetics, in which the rate of absorption from the lung intothe serum is much slower than the rate of elimination from the serum.

Example 17 Mass-Balance: Combined Lung-Derived Fractions

To analyze the mass-balance at different time points, the combinedamounts of material recovered in the lung-derived fractions werecalculated. FIG. 18 shows the combined amounts recovered represented bystacked histograms demonstrating the relative distribution among thecompartments measured at each time point.

FIGS. 18A and 18B demonstrate that for all the PEG-FITC molecules, themajority of the delivered dose is recovered at 10 min post-dose. Forsodium fluorescein, less than 50% of the dose given is recovered,indicating that this molecule, in the absence of conjugated PEG, israpidly absorbed from the lung. Although the serum compartment is notrepresented here, the concentrations in serum were so low that theamount in serum would not constitute a significant portion of the totaldose delivered, and thus would not alter these profiles.

Two features stand out among the graphs in FIGS. 18A and 18B. The firstis that at later time-points, the mass balance is not achieved, —i.e., asignificant portion of the delivered dose is not recovered. Serum datais so low as to preclude the serum or blood from containing the balanceof the delivered dose. Instead, the most likely explanation is that thePEG-FITC has been rapidly cleared by the kidneys, as discussedpreviously. The second notable feature is that the smaller molecularweight PEGs (0.55K and 1K) are fully cleared from all lung-derivedcompartments. The larger PEGs, by contrast, are not fully cleared evenby 48 h, and for the largest sizes (5K and 20K) between 25-25% of thedose remains in the combined lung-derived compartments (the BAL cellsand the residual lung tissue) even 7 days post-dose.

FIGS. 19A and 19B show the amount remaining in the combined lungdepartments (from FIGS. 18A and 18B) as concentration-time curves foreach PEG-FITC species. The curves closely resemble those in FIG. 12representing elimination of PEG-FITC from the BAL, and the half-livesfor these exponential decay curves are very close to those seen for BAL.Thus the main factor contributing to elimination from the entire lung isthe rate of elimination from the BAL.

The only species for which the elimination profile differs significantlybetween the BAL and the combined lung fractions is the 3.4K PEG-FITC. Asdiscussed above, this may relate to the “dumbbell” nature of themolecule which could alter its lung retention properties.

The PEG-FITC that is eliminated from the lung is most probably absorbeddirectly into the systemic circulation through the pulmonary epithelium.Alternative explanations for its elimination would be clearance in thelung by metabolic degradation or removal by the mucociliary escalator,leading to oral ingestion. It is highly unlikely that either of theseprocesses would exhibit the kinetics displayed here. If absorption intothe systemic circulation does indeed underlie the elimination from theBAL, then the rates of elimination should either be equal or close tothe rate of absorption into the systemic circulation.

Example 18 In Vitro Studies and Log P Data for PEG-FITC Conjugates

In vitro cell-based experiments (in a Calu-3 assay) were performed forPEG-FITC conjugates. The Calu-3 cells were cultured with an apicalair-interface to mimic the in vivo epithelium. The absorption datademonstrates similar rank order of permeability rates to data from invivo studies described above. FIG. 20 illustrates the permeability rateplotted as function of molecular weight in Calu-3 studies. FIG. 21illustrates cell-based permeability plotted versus the in vivoabsorption rate. The data shows that there is a good in vivo-in vitrocorrelation for PEG-FITC conjugates.

The relationship between log P and PEG size for PEG-FITC conjugates wasdetermined using a potentiometric titration method using a Sirius GLpKainstrument. FIG. 22 shows that addition of PEG causes a significantshift in log P to a more negative value. Interestingly, no specificrelationship between MW and PEG and log P is observed in the molecularweight range of 0.55 to 20 KDa.

This increasing hydrophilicity by PEG conjugation, together with the invivo absorption and in vitro permeability data, are consistent with amodel of absorption in which a hydrophobic molecule, followingPEGylation, moves from a predominantly transcellular route to apredominantly paracellular one. The hydrophilic nature of the PEGylatedsmall molecule would cause it to travel preferentially via the aqueousintercellular tight junctions. As the PEG chain length increases,however, the hydrodynamic radius grows and the diffusion rate of themolecule decreases, leading to a diminished rate of permeability throughthe tight junctions. The effective maximum permeability rate reached at5K PEG may represent the structural size limitation of the tightjunctions, such that “unwinding” likely occurs for the molecule to enterthe intercellular pore. Such an “unwinding” process might thereforerepresent the rate-limiting step for PEG molecules greater than 3.4K.

Example 19 Elimination of PEG and PEG-FITC from the Lung

Polydisperse PEG having a weight average molecular weight of 2 KDa wasused to study elimination of PEG and PEG-FITC from the lung. The studieswere performed in accordance with the procedures in Example 12. FIG. 23shows almost identical elimination from lung for 2K PEG and 2K PEG-FITC,suggesting that PEG properties dominate those of the small molecule atthis size of PEG. FIG. 24 shows that increasing the dose of 2K PEG10-fold does not significantly alter the elimination rate from the lung.This supports the claim for passive transport of PEG across theepithelium, since the hallmark of passive transport is that permeabilityrates are independent of concentration (and are therefore notsaturated). In vitro permeability in Calu-3 cells shows similar ratesfor free 2K PEG and 2K PEG-FITC, supporting the notion that PEGdominates the properties of the PEG-small molecule conjugate. The invitro permeability is shown in Table IX below.

TABLE IX Permeability of molecules from lung (rat IT) Elimination fromCell-based lung (rat IT) Molecule Permeability (cm/s) (hr−1) SodiumFluorescein 3.4E−07 ± 0.4E−07 1.38 Cipro HCl 8.6E−07 ± 0.1E−07 10.24 1KPEG-FITC 5.4E−08 ± 0.6E−08 0.139 1K PEG-Cipro 7.4E−08 ± 0.2E−08 0.216 2KPEG 2.3E−08 ± 0.8E−08 0.100 2K PEG-FITC 4.7E−08 ± 0.2E−08 0.097

Example 20 PEG-CIPRO Studies

1 KDa and 5 KDa polydisperse PEG molecules were conjugated tociprofloxacin (CIPRO). In vivo absorption data was collected through ratstudies, in which, following IT administration to rats, whole lungs wereexcised and analyzed for test article concentration. The studies showedthat addition of 1K PEG to CIPRO causes significant decrease inabsorption across pulmonary epithelium, evident from a decrease indisappearance from the lung, as illustrated in FIG. 25, and concomitantdecrease in rate of appearance in plasma, as illustrated in FIG. 26. Asshown in FIG. 27, the t_(1/2) elimination was about 4 minutes with CIPROalone and about 3.2 hours with the 1K PEG-CIPRO conjugate (FIG. 26).

This data shows that the elimination from the lung is paralleled byappearance in plasma, thus confirming the process of absorption throughthe pulmonary epithelium. The increased T_(max) of 1K-PEG-CIPRO reflectsthe slower absorption, resulting in a longer time taken to reach theC_(max). The decreased C_(max) of the 1K-PEG-CIPRO compared with CIPROmost likely arises predominantly from the decreased rate of absorption.Although the elimination from serum may be slightly decreased byPEGylation, the clearance through the kidneys would still be expected tobe fairly rapid. PEG linkage is via thioester bond which can hydrolyze,although data show this to be very slow, and the data presented derivesfrom the full PEG-CIPRO species detected by HPLC.

The CIPRO data, together with the free PEG and PEG-FITC data, supportthe claim that conjugation of PEG (≧1K) to a small molecule results indominance of the physico-chemical characteristics by PEG such that theconjugate essentially behaves like PEG. Thus the pharmacokineticbehaviour of the small molecule can be (predictably) controlled byconjugation to PEG of sizes ≧1K.

Example 21 Monodisperse PEG-Triamcinolone Molecules

In order to determine whether PEGylated compounds can cross a lipidbilayer and enter a cell, various monodisperse PEGS were conjugated tothe corticosteroid tiamcinolone (TA). TA crosses the membrane rapidly sothat any slowing effect of PEG conjugation could easily be measured.

To determine the location and movement of the PEGylated steroid,functional assays were planned which required the PEG moieties to beconjugated at sites in the steroid that would not inactivate themolecule. For corticosteroids, the 21′OH position is known to beessential for biological activity, rendering this site unsuitable forconjugation. However, modification of the hydroxyls at positions 16 and17 allows retention, sometimes improvement, of biological activity, asdemonstrated by the acetonide derivative of triamcinolone, formed by areaction between acetone and a 1,2-diol. Based on this, it was reasonedthat the steroid triamcinolone could be reacted with the mPEG aldehydeinstead of a ketone to yield an acetal with the structure shown below.The conjugates containing the 3-mer PEG or 7-mer PEG were termedPEG-3-TA or PEG-7-TA respectively. The resulting molecular weights ofPEG-3-TA and PEG-7-TA are 611 Da and 787 Da respectively. The structuresof the parent triamcinolone and triamcinolone acetonide are depicted inbelow in C and D respectively.

A is the chemical structure of PEG-TA showing conjugation of PEG moietyat 16 and 17 positions of the steroid ring. B is the structure of theundesired “contaminant” showing attachment of the PEG at the 21′OHposition, which will render this compound pharmacologically inactive. Cis the structure of the parent steroid triamcinolone. D is the structureof triamcinolone acetonide, demonstrating modification at the 16 and 17hydroxyls similar to that in PEG-TA.

For PEG-3-TA, the resulting reaction generated products in which thedesired structure (A) constituted 95% of the final reaction product withthe remaining 5% comprising the isomer depicted in B. For PEG-7-TA, therespective proportions were 90% and 10%. Since the isomeric contaminantcontains a covalent linkage at the 21-OH position, this molecule will beinactive and thus not contribute to the overall biological activity.HPLC analysis demonstrated that there was no unreacted parenttriamcinolone in the purified reaction product.

Example 22 Nuclear Translocation Assay of PEG-TA

To determine whether PEGylated steroids can enter cells, and to comparetheir potency with the parent steroid triamcinolone, cells expressingthe human glucocorticoid receptor (hGR) were incubated with increasingconcentrations of TA, PEG-3-TA and PEG-7-TA, and the subcellularlocation of the hGR determined using immunocytochemistry andfluorescence microscopy.

Chinese Hamster Ovary (CHO) cells were plated on glass coverslips in24-well plates, and transfected with DNA encoding the hGR using 0.25 μgDNA per well. One day following transfection, cells were incubated withincreasing concentrations, from 1 nM to 10 μM, of TA, PEG-3-TA orPEG-7-TA, overnight at 37° C.

Stock solutions of steroids were prepared at 2 mM (TA) or 1 mM(PEG-3-TA, PEG-7-TA) in ethanol. Each test compound was diluted to 10 μMinto culture medium supplemented with dextran-coated charcoal treatedserum (DCCS) (final 5% serum), and then further diluted to the finalconcentrations. This DCCS was employed to minimize background effectscaused by steroids in the culture medium. Dilutions of steroid werecalculated to ensure that cells were not exposed to ethanolconcentrations greater than 1% (v/v). Previous studies using 1% ethanolconfirmed that the solvent alone had no effect on the location of thehGR. Control, untreated cells were incubated with a 1:100 dilution ofCa²⁺/Mg²⁺-free PBS.

Following treatment with test compounds, cells were fixed in 3.7% (v/v)formaldehyde/PBS and permeabilized with 1% (v/v) Triton-X-100 inblocking buffer. Subcellular localization of the transfected hGR wasdetermined by immunocytochemistry using an anti-GR antibody and aFITC-conjugated secondary antibody. Cells were counterstained with DAPIwhich binds the nuclei of all cells, transfected and untransfected. hGRlocalization was detected using fluorescence microscopy, and imagestaken with a CCD high resolution digital camera using multidimensionalchannel recording to allow acquisition of overlapping images fromdifferent fluorescence wavelengths.

FIG. 28 shows a dose-dependent nuclear translocation of the hGR inducedby the three test compounds, TA, PEG-3-TA and PEG-7-TA, with a cleardifference in potency between the compounds. The top panels show controlsamples containing untreated cells. The green stain spread across thewhole cell is characteristic of cytosolic staining, with the nucleussomewhat discernible in the centre of the cell, and demonstrates that inthe basal state, the hGR is almost entirely cytosolic.

In the presence of 1 nM TA, the hGR localizes predominantly in thenucleus, with a very low degree of staining remaining in the cytosol.Complete translocation to the nucleus occurs with TA concentrations atand above 10 nM. By contrast, the PEGylated forms of triamcinolone donot alter the hGR distribution at the lowest concentration. ForPEG-3-TA, nuclear staining of the hGR begins to occur at 10 nM, and at 1μM, all the transfected hGR detected is present in the nucleus. ForPEG-7-TA, the translocating effect only begins to appear at 100 nM andis more clearly seen at 1 μM. At 10 μM, PEG-7-TA causes completetranslocation of the transfected hGR from the cytosol to the nucleus.

The data show clearly that the PEGylated forms of triamcinolone arecapable of inducing nuclear translocation of the hGR, albeit with muchreduced efficiency compared with the parent steroid. This demonstratesthat these compounds are capable of entering cells and of binding thereceptor. Although this assay is not sufficiently quantitative tomeasure accurately the relative potency of the three compounds, it isclear that the order of potency is TA>PEG-3-TA>PEG-7-TA.

Example 23 Glucocorticoid Receptor Reporter Assay of PEG-TA

To quantitate the effect of PEGylation on the ability of the steroid toenter a cell and elicit a response, a glucocorticoid receptor reporterassay was employed. In this assay, treatment of cells with ligand leadsto receptor-mediated expression of the firefly luciferase gene. Thelevel of gene expression is quantitated by an enzymatic luciferaseassay, and the amount of light produced is directly proportional to theexpression level of the luciferase gene.

The results from the nuclear translocation assay demonstrate thatconjugation of PEG at the 16- and 17-OH positions of the steroid ringimpair, but do not prevent the drug from entering cells. Since TAAcontains modifications at the 16 and 17 positions, it was chosen as themore appropriate comparator for PEG-TA in this quantitative assay.

Experimental Details

For these experiments, the Promega Dual Luciferase Reporter kit wasemployed. This involves a firefly luciferase reporter gene under thecontrol of the Glucocorticoid Response Element (GRE-Luc), co-transfectedinto COS-7 cells with a gene encoding the hGR and a control vectorcontaining a Renilla luciferase reporter gene under no promoter orenhancer elements (pRL-Null). The firefly luciferase gene is expressedonly upon binding of a ligand-bound receptor to the GRE-Luc enhancer.The Renilla luciferase gene, by contrast, is constitutively expressedand can be assayed independently from the firefly luciferase, thusproviding a control for variation in transfection efficiencies betweensamples. This is based on the commonly accepted observation that upontransfection with multiple plasmids, cells will generally either take upall the plasmids or no plasmid. Thus although transfection efficiencyand cell numbers can vary between wells, cells transfected with thefirefly luciferase gene will also contain the Renilla luciferase geneand the activity of the former can be normalized to that of the latter.

Cos-cells were plated in 96-well microplates and transfected with therelevant plasmids using standard procedures; all incubations and assayswere performed in triplicate in these plates using medium containingDCCS, as described in section 8.3.1. Twenty-four hourspost-transfection, cells were incubated with different concentrations ofsteroid for 20 hours. Cells were then lysed, and the expression levelsof firefly and Renilla luciferase were determined by sequentialenzymatic assays provided by the Promega Dual Luciferase Reporter Assaykit. The resulting luminescence was detected using the Tecan Genios ProMicroplate reader, and data were analyzed using GraphPad Prism 4.0software.

Readings obtained in the absence of steroid represent “basal” backgroundlevels of luciferase expression. All readings in the presence of steroidare expressed as a ratio compared with this, and termed “fold increaseover basal.” Values obtained for firefly luciferase were normalized tothose obtained for Renilla luciferase in the corresponding well in orderto compensate for differences in transfection efficiency or cell number.

In time-course experiments (e.g. FIG. 30), transfected cells wereincubated with four different concentrations of steroid for increasingtime periods. The time points at which test articles were applied to thecells were staggered to enable all reactions to terminate at the sametime, thus allowing simultaneous assaying of all samples in a singlemicroplate.

Results

Comparison of EC₅₀ values for TAA, PEG-3-TA, and PEG-7-TA revealed amarked difference in potency between the native and the PEGylatedsteroids. An EC₅₀ value represents the concentration of drug thatproduces a half-maximal response, and serves as the standard measure ofcomparing potencies of different compounds. Thus, a higher EC₅₀ valuesignifies a lower potency. FIG. 29 displays a representative experiment,showing a difference in EC₅₀ of 3 orders of magnitude between TAA andPEG-3-TA or PEG-7-TA. The figure demonstrates that the PEGylatedsteroids operate with a slightly lower efficacy (maximal response) but adramatically reduced potency (EC₅₀) at these receptors. Comparative EC₅₀values from a number of experiments are recorded in Table X, and revealdifferences in potency from TAA of approximately 1300-fold and 5500-foldfor PEG-3-TA and PEG-7-TA respectively.

To determine whether a similar difference is observed with the parentsteroid, TA, which contains no modification at the 16- and 17-OHpositions, the luciferase reporter assay was performed on TA.

TA displayed differences in EC₅₀ values of 150-fold and 630-foldrespectively when compared with PEG-3-TA and PEG-7-TA, and wasapproximately 9-fold less potent than TAA. These data correlate well tothe results obtained in the nuclear translocation assay, in which anapproximate 2 orders of magnitude difference was noted between thePEGylated and parent forms of the steroid, and an additional differencefound between the shorter (3-mer) and longer (7-mer) PEGylatedderivatives.

To confirm that experiments were performed at equilibrium, time courseexperiments were performed. FIG. 30 demonstrates that by 20 hincubation, receptor activation values had reached their maximal levelsand did not vary significantly for up to 48 h incubation, regardless ofthe concentration used. At early time-points, i.e. less than 6 h,submaximal activation was observed. This time course most likelyreflects the lag time for the RNA transcription and protein synthesisthat is required for the luciferase enzymes to be generated.

Verification that the PEG-TA derivatives were also assayed atequilibrium is found in the experiment (Luc-21) detailed in Appendix I,in which plates prepared in parallel were assayed after incubation withsteroid for 24 or 48 h. The similar results produced in both platesconfirm that reactions had reached equilibrium.

Example 24 Glucocorticoid Receptor Binding Assay of PEG-TA

To determine what component of the differences in receptor activationare due to the effect of PEG on receptor binding, a cell-free in vitroGR binding assay was employed. The assay is based on competitiveinhibition binding of a fluorescently labeled high affinity ligand, andgenerates Ki values that can be compared between the test compounds. TheKi is a measure of the affinity of the binding interaction and derivesfrom the concentration of competing ligand that produces 50% inhibitionof maximal binding. Thus, as with EC_(5o) values, a higher Ki valuereflects a weaker binding affinity.

A commercially available GR binding assay based on fluorescencepolarization (FP) was utilized to compare the binding affinity of thePEGylated and native steroids (PanVera Glucocorticoid ReceptorCompetitor Assay). The assay is based on competitive inhibition andmeasures the ability of the test compound to compete with afluorescently labeled ligand (Fluormone™ GS1) for binding to a purifiedpreparation of the recombinant human glucocorticoid receptor.

In its free unbound form, the fluorescent ligand behaves as a smallmolecule with a high rotational velocity or “tumbling rate”, andcorrespondingly low ability to polarize fluorescence light (i.e. a lowFP). When bound to receptor, the ligand essentially becomes part of alarger molecule, with a slower rotational velocity and a higher FP.Addition of competing ligand, the test compound, results in adose-dependent decrease in FP as the fluorescent ligand is preventedfrom binding the receptor.

Standard competition assay procedures were employed. Briefly, serialdilutions of the test compound were incubated in triplicate wells of ablack 96-well microplate together with the fluorescent ligand, andreactions were initiated by addition of purified recombinant receptor.Assays were incubated in the dark for 2-4 h, and then polarizationvalues were read using the fluorescence polarization function of theTecan Genios Pro microplate reader.

FIG. 31 shows results from a representative experiment and reveals arelatively modest difference in binding affinity between TA and thePEG-TA derivatives. In contrast to the large difference in potencyobserved in the luciferase assay, the cell-free binding assay producesdifferences in binding affinity of less than one order of magnitude.Average data from replicate experiments, detailed in Table X, produce a4.2-fold and an 8.8-fold increase in Ki values for PEG-3-TA and PEG-7-TArespectively compared with TAA.

Interestingly, TA displayed an even lower binding affinity, with a Kiapproximately 20-fold lower than that of TAA. The Ki value fordexamethasone was comparable to that of TAA, whereas that of a relatedcorticosteroid, budesonide, appeared to be slightly lower.

TABLE X Assay REQUIRING entry to cell Luciferase assay TAA TA* PEG-3-TAPEG-7-TA EC₅₀ 0.062 ± 0.011 nM 0.545 nM 81.7 ± 9.0 nM 343.2 ± 54.2 nMFold difference 1 8.8 1316 5533 vs TAA Assay INDEPENDENT of entry tocell Binding assay TAA TA** PEG-3-TA PEG-7-TA Ki 2.2 ± 0.5 nM 45.5 ±21.1 nM 9.4 ± 2.4 nM 19.6 ± 5.1 nM Fold difference 1 20.3 4.2 8.8 vs TAA*(n = 1) **(n = 2) Data shown are EC₅₀ and Ki values ± standard error.TA values are based on 1 or 2 experiments as indicated. All other valuesare based on 5 or more experiments. Further details for these data canbe found in Appendix I.

Except where otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

1-38. (canceled)
 39. A method of preparing a monodisperse oligo(ethylene glycol) reagent composition, comprising: reacting ahalo-terminated oligo (ethylene glycol) having (m) monomer subunits anda methoxy end-capping group with a first hydroxyl-terminated oligo(ethylene glycol) having (n) monomer subunits, wherein the firsthydroxyl-terminated oligo (ethylene glycol) structure isHO—(CH₂CH₂O)_(n)—H, under conditions effective to displace said halogroup to thereby foi in an oligo (ethylene glycol) having (m)+(n)monomer subunits (OEG_(m+n)), wherein reacting the halo-terminatedoligo(ethylene glycol) with the first hydroxyl-terminated oligo(ethylene glycol) comprises adding a 1:1 molar ratio of thehalo-terminated oligo(ethylene glycol) to the first hydroxyl-terminatedoligo (ethylene glycol) in a solution; converting the terminal hydroxylgroup of OEG_(m+n) into a halo group, —X, to form OEG_(m+n)-X; andreacting OEG_(m+n)-X with a second hydroxyl-terminated oligo (ethyleneglycol) having (n) monomer subunits, wherein the secondhydroxyl-terminated oligo structure is HO—(CH₂CH₂O)_(n)—H, underconditions effective to displace said halo group to thereby form anoligo(ethylene glycol) having (m)+2(n) monomer subunits (OEG_(m+2n)),wherein reacting the OEG_(m+n)-X with the second hydroxyl-terminatedoligo (ethylene glycol) comprises adding a 1:1 molar ratio of theOEG_(m+n)X to the second hydroxyl-terminated oligo (ethylene glycol) ina solution; wherein (m) and (n) each independently range from 1-10, andwherein (OEG_(m+2n)) corresponds to the structureCH₃—O—(CH₂CH₂O)_(m+2n)H, and is present as part of a monodispersecomposition of CH₃—O—(CH₂CH₂O)_(m+2n)H.
 40. A method of preparing amonodisperse oligo (ethylene glycol) reagent composition, comprising:reacting a halo-terminated oligo (ethylene glycol) having (m) monomersubunits and a methoxy end-capping group with a firsthydroxyl-terminated oligo (ethylene glycol) having (n) monomer subunits,wherein the first hydroxyl-terminated oligo (ethylene glycol) structureis HO—(CH₂CH₂O)_(n)—H, under conditions effective to displace said halogroup to thereby form an oligo (ethylene glycol) having (m)+(n) monomersubunits (OEG_(m+n)), wherein reacting the halo-terminatedoligo(ethylene glycol) with the first hydroxyl-terminated oligo(ethylene glycol) comprises adding a 1:1 molar ratio of thehalo-terminated oligo(ethylene glycol) to the first hydroxyl-terminatedoligo (ethylene glycol) in a solution; converting the terminal hydroxylgroup of OEG_(m+n) into a halo group, —X, to form OEG_(m+n)-X; reactingOEG_(m+n)-X with a second hydroxyl-terminated oligo (ethylene glycol)having (n) monomer subunits under conditions effective to displace saidhalo group to thereby form an oligo(ethylene glycol) having (m)+2(n)monomer subunits (OEG_(m+2n)), wherein OEG_(m+2n) possesses a primary orsecondary hydroxyl group, wherein reacting the OEG_(m+n)-X with thesecond hydroxyl-terminated oligo (ethylene glycol) comprises adding a1:1 molar ratio of the OEG_(m+n)-X to the second hydroxyl-terminatedoligo (ethylene glycol) in a solution; converting said primary orsecondary hydroxyl group into a halo group, —X, to form OEG_(m+2n)-X;and reacting OEG_(m+2n)-X with a third hydroxyl-terminated oligo(ethylene glycol) having (n) monomer subunits under conditions effectiveto displace said halo group to thereby form an oligo (ethylene glycol)having m+3n monomer subunits (OEG_(m+3n)), wherein reacting theOEG_(m+2n)-X with the third hydroxyl-terminated oligo (ethylene glycol)comprises adding a 1:1 molar ratio of the OEG_(m+2n)-X to the thirdhydroxyl-terminated oligo (ethylene glycol) in a solution; wherein (m)and (n) each independently range from 1-10; and wherein (OEG_(m+3n))corresponds to the structure CH₃—O—(CH₂CH₂O)_(m+3n)H, and is present asa monodisperse composition of CH₃—O—(CH₂CH₂O)_(m+3n)H.
 41. The method ofclaim 39, wherein (m) ranges from 1-3 and (n) ranges from 2-6.
 42. Themethod of claim 40, wherein (m) ranges from 1-3 and (n) ranges from 2-6.43. The method of claim 39, wherein each reacting step is carried out inthe presence of a strong base.
 44. The method of claim 40, wherein eachreacting step is carried out in the presence of a strong base.
 45. Themethod of claim 39, wherein each of the halo groups is Br.
 46. Themethod of claim 40, wherein each of the halo groups is Br.